Muteins of hNGAL and related proteins with affinity for a given target

ABSTRACT

The present invention relates to novel muteins derived from human lipocalin 2 (hNGAL) and related proteins that bind a given non-natural ligand with detectable affinity. The invention also related to corresponding nucleic acid molecules encoding such a mutein and to a method for their generation. The invention further relates to a method for producing such a mutein. Furthermore, the invention is directed to a pharmaceutical composition comprising such a lipocalin mutein as well as to various uses of the mutein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.12/737,240, which is a National Stage application of PCT/EP2009/057925,filed Jun. 24, 2009, which claims the benefit of priority of U.S.provisional application No. 61/075,175, filed Jun. 24, 2008, the entirecontents of which are incorporated herein for all purposes.

FIELD OF THE INVENTION

The present invention relates to novel muteins derived from humanlipocalin 2 (hNGAL) and related proteins that bind a given non-naturalligand with detectable affinity. The invention also relates tocorresponding nucleic acid molecules encoding such a mutein and to amethod for their generation. The invention further relates a method forproducing such a mutein. Furthermore, the invention is directed to apharmaceutical composition comprising such a lipocalin mutein as well asto various uses of the mutein.

BACKGROUND OF THE INVENTION

The lipocalins are a diverse family of small and robust, secretoryproteins which serve for the transport or storage of poorly soluble orchemically sensitive vitamins, hormones, and metabolites (such asretinoids, fatty acids, cholesterols, prostaglandins, biliverdins,pheromones, tastants, and odorants) in many organisms (Åkerström et al.Eds. (2006), Lipocalins, Landes Bioscience, Georgetown, Tex.; Pervaiz,S., and Brew, K. (1987) FASEB J. 1, 209-214). Although they have, in thepast, been classified primarily as transport proteins, it is now clearthat the lipocalins fulfill a variety of physiological functions. Theseinclude roles in retinol transport, olfaction, pheromone signaling, andthe synthesis of prostaglandins. The lipocalins have also beenimplicated in the regulation of the immune response and the mediation ofcell homoeostasis (reviewed, for example, in Flower, D. R. (1996)Biochem. J. 318, 1-14 and Flower, D. R. et al. (2000) Biochim. Biophys.Acta 1482, 9-24).

The lipocalins share unusually low levels of overall sequenceconservation, often with sequence identities of less than 20%. In strongcontrast, their overall folding pattern is highly conserved. The centralpart of the lipocalin structure consists of a single eight-strandedanti-parallel β-sheet closed back on itself to form a continuouslyhydrogen-bonded β-barrel. One end of the barrel is sterically blocked bythe N-terminal peptide segment that runs across its bottom as well asthree peptide loops connecting the β-strands. The other end of theβ-barrel is open to the solvent and encompasses a target-binding site,which is formed by four peptide loops. It is this diversity of the loopsin the otherwise rigid lipocalin scaffold that gives rise to a varietyof different binding modes each capable of accommodating targets ofdifferent size, shape, and chemical character (reviewed, e.g., inFlower, D. R. (1996), supra; Flower, D. R. et al. (2000), supra, orSkerra, A. (2000) Biochim. Biophys. Acta 1482, 337-350).

Among the 10-12 members of the lipocalin family that are found in thehuman body, human neutrophil gelatinase-associated lipocalin (hNGAL)(Kjeldsen et al. (2000) Biochim. Biophys. Acta 1482, 272-283)—also knownas lipocalin 2 (Lcn2) or, more recently, dubbed siderocalin (Goetz etal. (2002) Mol. Cell 10, 1033-1043)—plays a role in the innate immunedefence against bacterial infections by scavenging Fe³⁺ ions bound tocertain bacterial siderophores.

Such siderophores are highly potent iron chelators which are secreted bypathogenic bacteria in response to limiting iron concentrations(Schaible & Kaufmann (2004). Nat. Rev. Microbiol. 2, 946-953), as theyhappen in the human body fluids, to allow iron uptake by specializedbacterial import systems (Braun & Braun (2002) Curr. Opin. Microbiol. 5,194-201; Fischbach et al. (2006) Nat. Chem. Biol. 2, 132-138). It seemsthat neutrophils release hNGAL at sites of infection as an antimicrobialstrategy. Indeed, the physiological relevance of hNGAL has beendemonstrated in corresponding knock-out mice, where this lipocalin wasshown to be essential in limiting the spreading of bacteria that rely onenterobactin-mediated iron import (Flo et al. (2004) Nature 432,917-921)

hNGAL (also termed Lcn2, SWISS-PROT Data Bank Accession Number P80188)is a 178 amino acid glycoprotein with strong binding activity towardsthe catecholate-type siderophore Fe³⁺.enterobactin (or enterochelin),which is characteristic for Escherichia coli (Raymond et al. (2003)Proc. Natl. Acad. Sci. USA 100, 3584-8). hNGAL is an abundant humanplasma protein, whose normal concentration is around 80 μg/L and canincrease up to ten-fold upon bacterial infections (Xu and Venge (2000)Biochim. Biophys. Acta 1482, 298-307), and its single N-linkedglycosylation site is dispensable for folding (Coles et al. (1999) J.Mol. Biol. 289, 139-157). Compared with other lipocalins, hNGAL exhibitsan unusually large pocket. Therein, a cluster of positively charged sidechains confers extraordinary affinity for the negatively charged ferricsiderophore, with a dissociation constant (K_(D)) of 0.4 nM (Goetz etal., supra), thus allowing effective competition with the bacterialuptake system. Ligand recognition by hNGAL is rather specific as thislipocalin also forms stable complexes with the chemically relatedbacillibactin from Bacillus anthracis (Abergel et al. (2006) Proc. Natl.Acad. Sci. USA 103, 18499-18503) and with carboxymycobactins fromMycobacterium tuberculosis (Holmes et al. (2005) Structure 13, 29-41), asiderophore type of similar size and shape. However, it does not bindpetrobactin, the siderophore that is crucial for virulence of B.anthracis (Abergel et al., supra), or C-glycosylated enterobactinanalogues such as the salmochelins produced by Salmonella spp. andKlebsiella pneumoniae (Fischbach et al., supra). Animal homologs tohuman Lcn2 are rat α₂-microglobulin-related protein (A2m; SWISS-PROTData Bank Accession Number P31052) and mouse 24p3/uterocalin (24p3;SWISS-PROT Data Bank Accession Number P11672).

Proteins that selectively bind to their corresponding targets by way ofnon-covalent interaction play a crucial role as reagents inbiotechnology, medicine, bioanalytics as well as in the biological andlife sciences in general. Antibodies, i.e. immunoglobulins, are aprominent example of this class of proteins. Despite the manifold needsfor such proteins in conjunction with recognition, binding and/orseparation of ligands/targets, almost exclusively immunoglobulins arecurrently used. The application of other proteins with definedligand-binding characteristics, for example the lectins, has remainedrestricted to special cases.

Rather recently, members of the lipocalin family have become subject ofresearch concerning proteins having defined ligand-binding properties.The PCT publication WO 99/16873 discloses polypeptides of the lipocalinfamily with mutated amino acid positions in the region of the fourpeptide loops, which are arranged at the end of the cylindrical β-barrelstructure encompassing the binding pocket, and which correspond to thosesegments in the linear polypeptide sequence comprising the amino acidpositions 28 to 45, 58 to 69, 86 to 99, and 114 to 129 of thebilin-binding protein of Pieris brassicae.

The PCT publication WO 00/75308 discloses muteins of the bilin-bindingprotein, which specifically bind digoxigenin, whereas the InternationalPatent Applications WO 03/029463 and WO 03/029471 relate to muteins ofthe human neutrophil gelatinase-associated lipocalin (hNGAL) andapolipoprotein D, respectively. In order to further improve and finetune ligand affinity, specificity as well as folding stability of alipocalin variant various approaches using different members of thelipocalin family have been proposed (Skerra, A. (2001) Rev. Mol.Biotechnol. 74, 257-275; Schlehuber, S., and Skerra, A. (2002) Biophys.Chem. 96, 213-228), such as the replacement of additional amino acidresidues. The PCT publication WO 2006/56464 discloses muteins of humanneutrophil gelatinase-associated lipocalin with binding affinity forCTLA-4 in the low nanomolar range.

The PCT publication WO 2005/19256 discloses muteins of tear lipocalinwith at least one binding site for different or the same target ligandand provides a method for the generation of such muteins of human tearlipocalin. According to this PCT application, certain amino acidstretches within the primary sequence of tear lipocalin, in particularthe loop regions comprising amino acids 7-14, 24-36, 41-49, 53-66,69-77, 79-84, 87-98, and 103-110 of mature human tear lipocalin, aresubjected to mutagenesis in order to generate muteins with bindingaffinities. The resulting muteins have binding affinities for theselected ligand (K_(D)) in the nanomolar range.

The lipocalin muteins disclosed in the above references are selected topreferentially bind large, proteinaceous target molecules and not smallmolecules. Thus, despite the progress made in this field, it would bedesirable to have hNGAL muteins that are specifically adapted to bindsmall molecules with high binding affinity, for example in the nanomolarrange. Such muteins would further improve the suitability of muteins ofhNGAL in diagnostic and therapeutic applications.

This object is accomplished by a mutein of hNGAL or of a related proteinhaving the features of the independent claims.

In a first aspect, the present invention provides a mutein derived froma protein selected from the group consisting of human neutrophilgelatinase-associated lipocalin (hNGAL), rat α₂-microglobulin-relatedprotein (A2m) and mouse 24p3/uterocalin (24p3), said mutein including atleast one mutated amino acid residue at any of the sequence positionscorresponding to the sequence positions 33, 36, 41, 52, 54, 68, 70, 79,81, 134, 136 and 138 of the linear polypeptide sequence of hNGAL, andwherein the mutein binds a given target with detectable affinity.

DETAILED DESCRIPTION OF THE INVENTION

In this context, it is noted that the invention is based on thesurprising finding that subjecting human neutrophilgelatinase-associated lipocalin (hNGAL), rat α₂-microglobulin-relatedprotein (A2m) and mouse 24p3/uterocalin (24p3) to mutagenesis at one ormore of these above-mentioned 12 sequence position provides for muteinsthat have a sufficiently affine binding to pre-defined target with lowmolecular weight.

In this context, it is also noted that in modern medicine, compounds oflow molecular weight such as metal-chelate complexes play an increasingrole for medical purposes, for example, the purposes of radio-immunotherapy (RIT)) and also for diagnostic purposes, for example in vivoimaging (Kenanova and Wu (2006) Expert Opin. Drug Deliv. 3, 53-70).Typically, for such a purpose, antibodies directed againsttumor-specific cell surface markers or peptides specific fordisease-related receptors have so far chemically conjugated to potentsynthetic chelating agents (Milenic et al. (2004) Nat. Rev. Drug Discov.3, 488-499), in particular DOTA(1,4,7,10-tetra-azacylcododecane-N,N′,N″,N′″-tetraacetic acid) and DTPA(diethylenetriamine pentaacetic acid) or their derivatives, which arethen charged with radionuclides of the rare earth elements such as Y³⁺or Lu³⁺ or similar trivalent metal ions, e.g. In³⁺ or Bi³⁺. Tworadionuclide-conjugated antibodies directed against CD20, Zevalin® andBexxar®, have been approved for the therapy of non-Hodgkin's lymphomaand many antibodies and their fragments are currently subject to proteinengineering for improved pharmacokinetics and tumor targeting (Kenanovaand Wu, supra).

A major obstacle of humanized antibodies for nuclear medicine is thelong circulation time, which leads to low contrast for imaging andlimited tumor specificity during RIT. To circumvent this problem,so-called pre-targeting strategies have been developed, where thetumor-targeting antibody is uncoupled from the chelated radionuclide(Chang et al. (2002) Mol. Cancer Ther. 1, 553-563). This enables theslow process of antibody localization and clearance from circulation inthe first stage, prior to the fast and specific delivery of the smallmolecule radioactive payload in the second stage. Initially,antibody-streptavidin conjugates were applied in conjunction withbiotinylated radionuclide chelates and, later, bispecific antibodiestogether with epitope peptide-conjugated chelate complexes. Moreover,monoclonal antibodies were developed which can directly bind the metalchelate (Le Doussal et al. (1990) Cancer Res. 50, 3445-3452; Corneillieet al. (2003) J. Am. Chem. Soc. 125, 3436-3437; Corneillie et al. (2003)J. Am. Chem. Soc. 125, 15039-15048).

The ideal system, however, would be a small metal chelate-specificbinding protein, comprising a single polypeptide chain with robustfolding properties, which can simply be coupled to a targetingpeptide/protein module—e.g. a natural receptor ligand, an antibodyfragment (Kenanova and Wu, supra) or an alternative binding protein(Skerra (2007) Curr. Opin. Biotechnol. 18, 295-304; Skerra (2007) Curr.Opin. Mol. Ther. 9, 336-344)—using a gene fusion strategy. The muteinsof hNGAL, rat α₂-microglobulin-related protein (A2m) and mouse24p3/uterocalin (24p3) provide such a protein having high bindingaffinity for such small target molecules.

The term “human neutrophil gelatinase-associated lipocalin” or “hNGAL”or “lipocalin 2” or “Lcn2” as used herein to refer to the mature hNGALwith the SWISS-PROT Data Bank Accession Number P80188. The amino acidsequence of human neutrophil gelatinase-associated lipocalin is setforth in SEQ ID NO: 1. The terms “rat α₂-microglobulin-related protein”or “A2 m” and “mouse 24p3/uterocalin” or “24p3” as used in the presentapplication refer to mature A2m or 24p3 with the SWISS-PROT Data BankAccession Numbers P31052 and P11672, respectively.

The given target may be any desired non-natural target/ligand. The term“non-natural ligand” refers to any compound, which does not bind tonative mature hNGAL under physiological conditions. The target (ligand)may be any chemical compound in free or conjugated form which exhibitsfeatures of an immunological hapten, for example, a small organicmolecule, such as a metal-chelating agent, or a peptide, for example of2 to about 25 or about 30 or about 35 amino acids length (see below).

The term “organic molecule” or “small organic molecule” as used hereindenotes an organic molecule comprising at least two carbon atoms, butpreferably not more than 7 or 12 rotatable carbon bonds, having amolecular weight in the range between 100 and 2000 Dalton, preferablybetween 100 and 1000 Dalton, and optionally including one or two metalatoms.

The term “peptide” as used herein with reference to a target moleculerefers to a dipeptide or an oligopeptide with of 2-40, 2-35, 2-30, 2-25,2-20, 2-15 or 2-10 amino acid residues. The peptide may be a naturallyoccurring or synthetic peptide and may comprise—besides the 20 naturallyoccurring L-amino acids—D-amino acids, non-naturally occurring aminoacids and amino acid analogs.

An hNGAL mutein (or the mutein of rat α₂-microglobulin-related protein(A2m) and mouse 24p3/uterocalin (24p3) of the invention may comprise thewild type (natural) amino acid sequence outside the mutated amino acidsequence positions. On the other hand, the lipocalin muteins disclosedherein may also contain amino acid mutations outside the sequencepositions subjected to mutagenesis as long as those mutations do notinterfere with the binding activity and the folding of the mutein. Suchmutations can be accomplished very easily on DNA level using establishedstandard methods (Sambrook, J. et al. (1989) Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). Possible alterations of the amino acid sequenceare insertions or deletions as well as amino acid substitutions. Suchsubstitutions may be conservative, i.e. an amino acid residue isreplaced with a chemically similar amino acid residue. Examples ofconservative substitutions are the replacements among the members of thefollowing groups: 1) alanine, serine, and threonine; 2) aspartic acidand glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine;5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine,tyrosine, and tryptophan. One the other hand, it is also possible tointroduce non-conservative alterations in the amino acid sequence. Inaddition, instead of replacing single amino acid residues, it is alsopossible to either insert or delete one or more continuous amino acidsof the primary structure of hNGAL as long as these deletions orinsertion result in a stable folded/functional mutein.

Such modifications of the amino acid sequence include directedmutagenesis of single amino acid positions in order to simplifysub-cloning of the mutated lipocalin gene or its parts by incorporatingcleavage sites for certain restriction enzymes. In addition, thesemutations can also be incorporated to further improve the affinity of alipocalin mutein for a given target. Furthermore, mutations can beintroduced in order to modulate certain characteristics of the muteinsuch as to improve folding stability, serum stability, proteinresistance or water solubility or to reduce aggregation tendency, ifnecessary. For example, naturally occurring cysteine residues may bemutated to other amino acids to prevent disulphide bridge formation.However, it is also possible to deliberately mutate other amino acidsequence position to cysteine in order to introduce new reactive groups,for example for the conjugation to other compounds, such as polyethyleneglycol (PEG), hydroxyethyl starch (HES), biotin, peptides or proteins,or for the formation of non-naturally occurring disulphide linkages.Exemplary possibilities of such a mutation to introduce a cysteineresidue into the amino acid sequence of an hNGAL mutein include theintroduction of a cysteine (Cys) residue at at least one of the sequencepositions that correspond to sequence positions 14, 21, 60, 84, 88, 116,141, 145, 143, 146 or 158 of the wild type sequence of hNGAL. Thegenerated thiol moiety at the side of any of the amino acid positions14, 21, 60, 84, 88, 116, 141, 145, 143, 146 and/or 158 may be used toPEGylate or HESylate the mutein, for example, in order to increase theserum half-life of a respective hNGAL mutein.

In one embodiment of the invention, the mutein includes mutated aminoacid residues at at least any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or all 12of the sequence positions corresponding to the sequence positions 33,36, 41, 52, 54, 68, 70, 79, 81, 134, 136 and 138 of the linearpolypeptide sequence of hNGAL.

In a further embodiment of the invention, the mutein further includes atleast one mutated amino acid residue at any of the sequence positionscorresponding to the sequence positions 42, 48, 49, 55, 75, 77, 80 and127 of the linear polypeptide sequence of hNGAL. Such a mutein may, forexample, include at least 9 mutated amino acid residues at any of thesequence positions corresponding to the sequence positions 33, 36, 41,42, 48, 49, 52, 54, 55, 68, 70, 75, 77, 79, 80, 81, 127, 134, 136 and138 of the linear polypeptide sequence of hNGAL. In one embodiment ofthe present invention, the mutein includes mutated amino acid residuesat at least any 10, 14, 15 or all 20 of the above-listed sequencepositions. The mutein may further comprise at least one mutated aminoacid residue at any of the sequence positions corresponding to thesequence positions 43, 44, 46, 47, 50, 51, 59, 65, 78, 86, 87, 98, 99,103, 107, 110 and 111 of the linear polypeptide sequence of hNGAL. Sucha mutein may, for example, include at least 9 mutated amino acidresidues at any of the sequence positions corresponding to the sequencepositions 33, 36, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 54, 55,59, 65, 68, 70, 75, 77, 78, 79, 80, 81, 86, 87, 98, 99, 103, 107, 110,111, 127, 134, 136 and 138 of the linear polypeptide sequence of hNGAL.

In one embodiment of the present invention, the mutein includes mutatedamino acid residues at at least any 10, 14, 15, 20, 22, 24, 26, 28, 29,30, 31, 32, 33, 35 or all 37 of the above-listed sequence positions.

In a still further embodiment of the present invention, the muteinincludes with respect to the mature hNGAL wild type amino acid sequenceadditional amino acid replacements at at least one of the sequencepositions that correspond to sequence positions 65, 71, 73, 74, 116, 125and 135 of the wild type sequence of hNGAL.

In still another embodiment, the muteins of the present invention mayfurther include one or more of the amino acid replacements selected fromthe group consisting of Glu28→His, Cys87→Ser, and Thr145→Ala.

The lipocalin muteins of the invention are able to bind the desiredtarget with detectable affinity, i.e. with a dissociation constant of atleast 200 nM. Preferred in some embodiments are lipocalin muteins, whichbind the desired target with a dissociation constant for a given targetof at least 100, 20, 1 nM or even less. The binding affinity of a muteinto the desired target can be measured by a multitude of methods such asfluorescence titration, competition ELISA or surface plasmon resonance(Biacore).

It is readily apparent to the skilled person that complex formation isdependent on many factors such as concentration of the binding partners,the presence of competitors, ionic strength of the buffer system etc.Selection and enrichment is generally performed under conditionsallowing the isolation of lipocalin muteins having, in complex with thedesired target, a dissociation constant of at least 200 nM. However, thewashing and elution steps can be carried out under varying stringency. Aselection with respect to the kinetic characteristics is possible aswell. For example, the selection can be performed under conditions,which favor complex formation of the target with muteins that show aslow dissociation from the target, or in other words a low k_(off) rate.Alternatively, selection can be performed under conditions, which favorfast formation of the complex between the mutein and the target, or inother words a high k_(on) rate.

An hNGAL mutein of the invention typically exists as monomeric protein.However, it is also possible that an inventive lipocalin mutein is ableto spontaneously dimerise or oligomerise. Although the use of lipocalinmuteins that form stable monomers may be preferred for someapplications, e.g. because of faster diffusion and better tissuepenetration, the use of lipocalin muteins that form stable homodimers ormultimers may be advantageous in other instances, since such multimerscan provide for a (further) increased affinity and/or avidity to a giventarget. Furthermore, oligomeric forms of the lipocalin mutein may haveslower dissociation rates or prolonged serum half-life.

According to one embodiment of the present invention, the mutein binds asmall organic molecule. The small organic molecule may be ametal-chelating agent or a pharmaceutical agent, such as a carboxy oramino group containing metal chelating-agent. Non-limiting examples forsuch chelating agents are ethylene-diamine-tetraacetic acid (EDTA),diethylenetriamine pentaacetic acid (DPTA),1,4,7,10-tetra-azacylcododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) orderivatives thereof such as2-methyl-6-(p-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (1B4M-DOTA),2-(p-isothiocyanatobenzyl)-5,6-cyclohexano-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate(CHX-DOTA),2-(p-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (C-DOTA) and1,4,7,10-Tetraaza-N-(1-carboxy-3-(4-nitrophenyl)propyl)-N′,N″,N′″-tris(aceticacid) cyclododecane (PA-DOTA) (see for example, Chappell, L, Synthesisand evaluation of novel bifunctional chelating agents based on1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid forradiolabeling proteins. Nuclear Medicine and Biology, Volume 30, Issue6, Pages 581-595), to name only a few illustrative examples. The DPTA,DOTA or derivative thereof may be complexed with a metal ion, which, forexample, is selected from the group consisting of yttrium (Y), terbium(Tb), indium (In), lutetium (Lu) and bismuth (Bi). The DTPA derivativemay, for example, be cyclohexyl-DTPA, including the diethylenetriaminepentaacetic acid (DTPA) series of amino acids described in U.S. Pat.Nos. 5,124,471, 5,286,850 and 5,434,287. Another example of chelatingagents which can be bound by muteins of the present invention arehydroxamic acid-based bifunctional chelating agents as described in U.S.Pat. No. 5,756,825. Another example is the chelating agent termedp-SCN-Bz-HEHA(1,4,7,10,13,16-hexaazacyclo-octadecane-N,N′,N″,N′″,N″″,N′″″-hexaaceticacid) (Deal et al., J. Med. Chem. 42: 2988, 1999), which is an effectivechelator of radionuclides such as 225Ac.

In this context, it is noted that it is possible using the presentinvention to generate a mutein that can bind to any chelating agent thatin turn complexes a given radionuclide. Characteristics such as physicaland chemical properties and the nature of the radiation are determinantsof the suitability of a radionuclide for therapy. Cytotoxicradionuclides may be divided into 3 groups of radiochemicals: halogens(iodine, ²¹¹At), metals (⁹⁰Y, ⁶⁷Cu, ²¹³Bi, ²¹²Bi), and transitionalelements (¹⁸⁶Re). Radionuclides can further be categorized into 4 typesof cytotoxic agents: pure β-emitters (⁶⁷Cu, ⁹⁰Y); α-emitters (²¹³Bi,²¹¹At) β-emitters that emit γ-radiation (¹⁷⁷Lu, ¹⁸⁶Re, ¹³¹I), and Augeremitters and radionuclides that decay by internal conversion, including¹²⁵I and ⁶⁷Ga. The use of muteins that bind to a chelating agent thatforms complexes with any of these radionuclides is contemplated in thepresent invention (cf., for example, Yuliya S. Jhanwar & ChaitanyaDivgi, Current Status of Therapy of Solid Tumors, Journal of NuclearMedicine Vol. 46 No. 1 (Suppl) 141S-150S, 2005).

Another purely illustrative example of a small molecule that can serveas given target are haptens such as norbornene haptens that have foundinterest as transition state analogue for the [4+2] Diels-Alder reaction(see for example, Xu et al., Science, 1999, Vol. 286, 2345-2348 orHilvert et al., J. Am. Chem. Soc. 1989, Vol. 111, 9261-9262). Such ahapten has been used in Example 16 to illustrate the suitability of thepresent invention to generate muteins with affinity towards everypossible small molecule against which also an immune response (thatmeans production of antibodies) can be generated. Thus, it is noted hereagain that the given target can be any hapten.

Alternatively, in another embodiment of the invention, the mutein of thepresent invention may bind a peptide, for example a peptide of 2-40,2-35, 2-30, 2-25, 2-20, 2-15, or 2-10 amino acids length. The peptidemay be a naturally occurring peptide, such as, for example, anangiotensin (angiotensin I-IV), a natriuretic peptide (ANP, BNP, CNP), avasopressin, an oxytocin or an opioid peptide (enkephalin, endorphin,dynorphin), or a synthetic peptide.

According to one embodiment of the present invention, the mutein ofhNGAL binds a chelating agent such as DOTA or cyclohexyl-DTPA with aK_(D) of 50 nM or less.

An hNGAL mutein of the invention that binds a chelating agent such asDOTA or cyclohexyl-DTPA may comprise with respect to the amino acidsequence of mature hNGAL at least at least 3, 4, 5, 6, 7, 8, 9, 10, 11,or 12 amino acid replacements selected from the group consisting ofVal33→Gln; Leu36→Arg; Ile41→Ala; Tyr52→Thr; Thr54→Gln; Ser68→Ala;Leu70→Arg; Trp79→Ala, Leu; Arg81→Met; Lys134→Ser; Thr136→Ser andTyr138→Leu. Generally, such a mutein binds cyclohexyl-DTPA with a K_(D)of 200 nM or less, 100 nM or less, 20 nM or less, or 1 nM or even lesswith a K_(D) in the picomolar range. Thus, the invention alsoencompasses hNGAL muteins that bind cyclohexyl-DTPA with a K_(D) of 900pM or less, 600 pM or less, 500 pM or less, 250 pM, 100 pM or less, 60pM or less or 40 pM or less. Suitable methods to determine K_(D) valuesof a mutein-ligand complex are known to those skilled in the art andinclude fluorescence titration, competition ELISA, calorimetric methods,such as isothermal titration calorimetry (ITC), and surface plasmonresonance. Examples for such methods are detailed below (See, e.g.,Examples).

In this context it is also noted that the complex formation between therespective mutein and its ligand is influenced by many different factorssuch as the concentrations of the respective binding partners, thepresence of competitors, pH and the ionic strength of the buffer systemused, and the experimental method used for determination of thedissociation constant K_(D) (for example fluorescence titration,competition ELISA or surface plasmon resonance, just to name a few) oreven the mathematical algorithm which is used for evaluation of theexperimental data.

Therefore, it is also clear to the skilled person that the K_(D) values(dissociation constant of the complex formed between the respectivemutein and its ligand) given here may vary within a certain experimentalrange, depending on the method and experimental setup that is used fordetermining the affinity of a particular lipocalin mutein for a givenligand. This means, there may be a slight deviation in the measuredK_(D) values or a tolerance range depending, for example, on whether theK_(D) value was determined by surface plasmon resonance (Biacore) or bycompetition ELISA.

In a specific embodiment of the invention, such a mutein furtherincludes with respect to the mature hNGAL wild type amino acid sequencean amino acid replacement selected from the group consisting ofLeu42→Pro; Pro48→Leu; Gln49→Leu; Ile55→Thr; Lys75→Met; Asp77→Glu;Ile80→Thr; and Ser127→Gln.

In one embodiment of the present invention, the hNGAL mutein bindingcyclohexyl-DPTA includes the amino acid substitutions: Val33→Gln;Leu36→Arg; Ile41→Ala; Tyr52→Thr; Thr54→Gln; Ser68→Ala; Leu70→Arg;Trp79→Ala or Leu; Arg81→Met; Lys134→Ser; Thr136→Ser; and Tyr138→Leu.Such a mutein may further include with respect to the mature hNGAL wildtype amino acid sequence one or more amino acid replacement selectedfrom the group consisting of Leu42→Pro; Pro 48→Leu; Gln49→Leu;Ile55→Thr; Lys75→Met; Asp77→Glu; Ile80→Thr; and Ser127→Gln.

In another embodiment, the hNGAL mutein includes with respect to themature hNGAL wild type amino acid sequence the amino acid replacements:

-   -   (a) Val33→Gln; Leu36→Arg; Ile41→Ala; Tyr52→Thr; Thr54→Gln;        Ser68→Ala; Leu70→Arg; Trp79→Ala; Arg81→Met; Lys134→Ser; and        Tyr138→Leu;    -   (b) Val33→Gln; Leu36→Arg; Ile41→Ala; Tyr52→Thr; Thr54→Gln;        Ser68→Ala; Leu70→Arg; Trp79→Leu; Arg81→Met; Lys134→Ser; and        Tyr138→Leu; or    -   (c) Val33→Gln; Leu36→Arg; Ile41→Ala; Tyr52→Thr; Thr54→Gln;        Ser68→Ala; Leu70→Arg; Trp79→Leu; Arg81→Met; Lys134→Ser;        Thr136→Ser; and Tyr138→Leu.

In such an embodiment, the mutein may further include with respect tothe mature hNGAL wild type amino acid sequence an amino acid replacementselected from the group consisting of Leu42→Pro, Pro48→Leu, Gln49→Leu,Ile55→Thr, Lys75→Met, Asp77→Glu, Ile80→Thr, and Ser127→Gln. The muteinmay further include with respect to the mature hNGAL wild type aminoacid sequence an amino acid replacement selected from the groupconsisting of Arg43→Pro, Glu44→Val, Glu44→Met, Lys46→Pro, Asp47→Glu,Lys50→Leu, Met51→Leu, Lys59→Arg, Asn65→Asp, Tyr78→His, Gly86→Ser,Ser87→Pro, Ser87→Phe, Lys98→Glu, Ser99→Asn, Leu103→Ile, Leu107→Phe,Val110→Met, and Val111→Ala.

In one embodiment of the present invention, the mutein comprises withrespect to the mature hNGAL wild type amino acid sequence the amino acidreplacements:

-   -   (a) Val33→Gln; Leu36→Arg; Ile41→Ala; Tyr52→Thr; Thr54→Gln;        Ser68→Ala; Leu70→Arg; Trp79→Ala; Ile 80→Thr; Arg81→Met;        Lys134→Ser; and Tyr138→Leu;    -   (b) Val33→Gln; Leu36→Arg; Ile41→Ala; Tyr52→Thr; Thr54→Gln;        Ser68→Ala; Leu70→Arg; Trp79→Leu; Ile 80→Thr; Arg81→Met;        Lys134→Ser; and Tyr138→Leu;    -   (c) Val33→Gln; Leu36→Arg; Ile41→Ala; Tyr52→Thr; Thr54→Gln;        Ser68→Ala; Leu70→Arg; Trp79→Leu; Ile 80→Thr; Arg81→Met;        Ser127→Gln; Lys134→Ser; and Tyr138→Leu;    -   (d) Val33→Gln; Leu36→Arg; Ile41→Ala; Tyr52→Thr; Thr54→Gln;        Ser68→Ala; Leu70→Arg; Trp79→Leu; Ile80→Thr; Arg81→Met;        Lys134→Ser; Thr136→Ser; and Tyr138→Leu;    -   (e) Val33→Gln; Leu36→Arg; Ile41→Ala; Tyr52→Thr; Thr54→Gln;        Ser68→Ala; Leu70→Arg; Trp79→Leu; Ile80→Thr; Arg81→Met;        Ser127→Gln; Lys134→Ser; Thr136→Ser; and Tyr138→Leu;    -   (f) Val33→Gln; Leu36→Arg; Ile41→Ala; Tyr52→Thr; Thr54→Gln;        Ser68→Ala; Leu70→Arg; Asp77→Glu; Trp79→Leu; Ile80→Thr;        Arg81→Met; Ser127→Gln; Lys134→Ser; Thr136→Ser; and Tyr138→Leu;    -   (g) Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro; Pro48→Leu;        Gln49→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr; Ser68→Ala;        Leu70→Arg; Lys75→Met; Asp77→Glu; Trp79→Leu; Ile80→Thr;        Arg81→Met; Ser127→Gln; Lys134→Ser; Thr136→Ser; and Tyr138→Leu;    -   (h) Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro; Arg43→Pro;        Glu44→Val; Lys46→Pro; Asp47→Glu; Pro48→Leu; Gln49→Leu;        Lys50→Leu; Met51→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr;        Ser68→Ala; Leu70→Arg; Lys75→Met; Asp77→Glu; Trp79→Leu; Ile        80→Thr; Arg81→Met; Ser127→Gln; Lys134→Ser; Thr136→Ser; and        Tyr138→Leu;    -   (i) Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro; Arg43→Pro;        Glu44→Val; Lys46→Pro; Asp47→Glu; Pro48→Leu; Gln49→Leu;        Lys50→Leu; Met51→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr;        Asn65→Asp; Ser68→Ala; Leu70→Arg; Lys75→Met; Asp77→Glu;        Trp79→Leu; Ile 80→Thr; Arg81→Met; Lys98→Glu; Val110→Met;        Ser127→Gln; Lys134→Ser; Thr136→Ser; and Tyr138→Leu;    -   (j) Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro; Arg43→Pro;        Glu44→Val; Lys46→Pro; Asp47→Glu; Pro48→Leu; Gln49→Leu;        Lys50→Leu; Met51→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr;        Asn65→Asp; Ser68→Ala; Leu70→Arg; Lys75→Met; Asp77→Glu;        Trp79→Leu; Ile 80→Thr; Arg81→Met; Gly86→Ser; Ser127→Gln;        Lys134→Ser; Thr136→Ser; and Tyr138→Leu;    -   (k) Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro; Arg43→Pro;        Glu44→Met; Lys46→Pro; Asp47→Glu; Pro48→Leu; Gln49→Leu;        Lys50→Leu; Met51→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr;        Asn65→Asp; Ser68→Ala; Leu70→Arg; Lys75→Met; Asp77→Glu;        Trp79→Leu; Ile 80→Thr; Arg81→Met; Gly86→Ser; Ser87→Pro;        Ser99→Asn; Leu107→Phe; Ser127→Gln; Lys134→Ser; Thr136→Ser; and        Tyr138→Leu;    -   (l) Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro; Arg43→Pro;        Glu44→Val; Lys46→Pro; Asp47→Glu; Pro48→Leu; Gln49→Leu;        Lys50→Leu; Met51→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr;        Lys59→Arg; Asn65→Asp; Ser68→Ala; Leu70→Arg; Lys75→Met;        Asp77→Glu; Trp79→Leu; Ile 80→Thr; Arg81→Met; Ser127→Gln;        Lys134→Ser; Thr136→Ser; and Tyr138→Leu;    -   (m) Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro; Arg43→Pro;        Glu44→Val; Lys46→Pro; Asp47→Glu; Pro48→Leu; Gln49→Leu;        Lys50→Leu; Met51→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr;        Asn65→Asp; Ser68→Ala; Leu70→Arg; Lys75→Met; Asp77→Glu;        Trp79→Leu; Ile 80→Thr; Arg81→Met; Ser87→Phe; Ser127→Gln;        Lys134→Ser; Thr136→Ser; and Tyr138→Leu; or    -   (n) Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro; Arg43→Pro;        Glu44→Val; Lys46→Pro; Asp47→Glu; Pro48→Leu; Gln49→Leu;        Lys50→Leu; Met51→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr;        Ser68→Ala; Leu70→Arg; Lys75→Met; Asp77→Glu; Tyr78→His;        Trp79→Leu; Ile 80→Thr; Arg81→Met; Leu103→Ile; Leu107→Phe;        Val111→Ala; Ser127→Gln; Lys134→Ser; Thr136→Ser; and Tyr138→Leu.

In any of the afore-mentioned embodiments, the mutein may furtherinclude with respect to the mature hNGAL wild type amino acid sequenceone, two or all three amino acid replacements selected from the groupconsisting of Glu28→His, Cys87→Ser, and Thr145→Ala.

The mutein may have an amino acid sequence selected from the groupconsisting of the sequences set forth in SEQ ID NOs. 2-10 and 28-34.

The hNGAL mutein binding cyclohexyl-DPTA may comprise, consistsessentially of or consist of any one of the amino acid sequences setforth in SEQ ID NOs.: 2-10 or 28-34 or a fragment or variant thereof. Inone embodiment, the mutein according to the invention comprises,consists essentially of or consists of the amino acid sequence set forthin SEQ ID NO: 8, 9, 10 or 28-34 or a fragment or variant thereof. Inthis regard, it is noted that all of the muteins disclosed herein can belinked, either N- or C-terminal to a affinity tag such as pentahistidinetag, a hexahistidine tag or a Streptag®. Thus, the present applicationencompasses also all explicitly and generic described muteins equippedwith such tags.

The term “fragment” as used in the present invention in connection withthe muteins of the invention relates to proteins or peptides derivedfrom full-length mature hNGAL that are N-terminally and/or C-terminallyshortened, i.e. lacking at least one of the N-terminal and/or C-terminalamino acids. Such fragments comprise preferably at least 10, morepreferably 20, most preferably 30 or more consecutive amino acids of theprimary sequence of mature hNGAL and are usually detectable in animmunoassay of mature hNGAL.

The term “variant” as used in the present invention relates toderivatives of a protein or peptide that comprise modifications of theamino acid sequence, for example by substitution, deletion, insertion orchemical modification. Preferably, such modifications do not reduce thefunctionality of the protein or peptide. Such variants include proteins,wherein one or more amino acids have been replaced by their respectiveD-stereoisomers or by amino acids other than the naturally occurring 20amino acids, such as, for example, ornithine, hydroxyproline,citrulline, homoserine, hydroxylysine, norvaline. However, suchsubstitutions may also be conservative, i.e. an amino acid residue isreplaced with a chemically similar amino acid residue. Examples ofconservative substitutions are the replacements among the members of thefollowing groups: 1) alanine, serine, and threonine; 2) aspartic acidand glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine;5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine,tyrosine, and tryptophan.

Also included in the scope of the present invention are the abovemuteins, which have been altered with respect to their immunogenicity.

Cytotoxic T-cells recognize peptide antigens on the cell surface of anantigen-presenting cell in association with a class I majorhistocompatibility complex (MHC) molecule. The ability of the peptidesto bind to MHC molecules is allele specific and correlates with theirimmunogenicity. In order to reduce immunogenicity of a given protein,the ability to predict which peptides in a protein have the potential tobind to a given MHC molecule is of great value. Approaches that employ acomputational threading approach to identify potential T-cell epitopeshave been previously described to predict the binding of a given peptidesequence to MHC class I molecules (Altuvia et al. (1995) J. Mol. Biol.249: 244-250).

Such an approach may also be utilized to identify potential T-cellepitopes in the muteins of the invention and to make depending on itsintended use a selection of a specific mutein on the basis of itspredicted immunogenicity. It may be furthermore possible to subjectpeptide regions which have been predicted to contain T-cell epitopes toadditional mutagenesis to reduce or eliminate these T-cell epitopes andthus minimize immunogenicity. The removal of amphipathic epitopes fromgenetically engineered antibodies has been described (Mateo et al.(2000) Hybridoma 19(6):463-471) and may be adapted to the muteins of thepresent invention.

The muteins thus obtained may possess a minimized immunogenicity, whichis desirable for their use in therapeutic and diagnostic applications,such as those described below.

For some applications, it is also useful to employ the muteins of theinvention in a conjugated form. Accordingly, the invention is alsodirected to lipocalin muteins which are conjugated to a conjungationpartner that may be selected from the group consisting of an enzymelabel, a colored label, a cytostatic agent, a label that can bephotoactivated and which is suitable for use in photodynamic therapy,haptens, digoxigenin, biotin, a chemotherapeutic metal, or achemotherapeutic metal, and colloidal gold, to name only a few evocativeexamples. The mutein may also be conjugated to an organic drug molecule.The conjugation can be carried out using any conventional couplingmethod known in the art.

In general, it is possible to label an hNGAL mutein described hereinwith any appropriate chemical substance or enzyme, which directly orindirectly generates a detectable compound or signal in a chemical,physical, optical, or enzymatic reaction. An example for a physicalreaction and at the same time optical reaction/marker is the emission offluorescence upon irradiation. Alkaline phosphatase, horseradishperoxidase or β-galactosidase are examples of enzyme labels (and at thesame time optical labels) which catalyze the formation of chromogenicreaction products. In general, all labels commonly used for antibodies(except those exclusively used with the sugar moiety in the Fc part ofimmunoglobulins) can also be used for conjugation to the muteins of thepresent invention. The muteins of the invention may also be conjugatedwith any suitable therapeutically active agent, e.g., for the targeteddelivery of such agents to a given cell, tissue or organ or for theselective targeting of cells, e.g., of tumor cells without affecting thesurrounding normal cells. Examples of such therapeutically active agentsinclude radionuclides, toxins, small organic molecules, and therapeuticpeptides (such as peptides acting as agonists/antagonists of a cellsurface receptor or peptides competing for a protein binding site on agiven cellular target). Examples of suitable toxins include, but are notlimited to pertussis-toxin, diphtheria toxin, ricin, saporin,pseudomonas exotoxin, calicheamicin or a derivative thereof, a taxoid, amaytansinoid, a tubulysin or a dolastatin analogue. The dolastatinanalogue may be auristatin E, monomethylauristatin E, auristatin PYE andauristatin PHE. Examples of cytostatic agent include, but are notlimited to Cisplatin, Carboplatin, Oxaliplatin, 5-Fluorouracil, Taxotere(Docetaxel), Paclitaxel, Anthracycline (Doxorubicin), Methotrexate,Vinblastin, Vincristine, Vindesine, Vinorelbine, Dacarbazine,Cyclophosphamide, Etoposide, Adriamycine, Camptotecine, CombretatastinA-4 related compounds, sulfonamides, oxadiazolines,benzo[b]thiophenessynthetic spiroketal pyrans, monotetrahydrofurancompounds, curacin and curacin derivatives, methoxyestradiol derivativesand Leucovorin. The lipocalin muteins of the invention may also beconjugated with therapeutically active nucleic acids such as antisensenucleic acid molecules, small interfering RNAs, micro RNAs or ribozymes.Such conjugates can be produced by methods well known in the art.

In one embodiment, the muteins of the invention may also be coupled to atargeting moiety that targets a specific body region in order to deliverthe inventive muteins to a desired region or area within the body. Oneexample wherein such modification may be desirable is the crossing ofthe blood-brain-barrier. In order to cross the blood-brain barrier, themuteins of the invention may be coupled to moieties that facilitate theactive transport across this barrier (see Gaillard P J, et al. (2005)International Congress Series. 1277, 185-198 or Gaillard P J, et al.(2005) Expert Opin Drug Deliv. 2(2), 299-309). Such moieties are forexample available under the trade name 2B-Trans™ (to-BBB technologiesBV, Leiden, NL). Other exemplary targeting molecules to which themuteins of the present invention may be coupled include antibodies,antibody fragments or lipocalin muteins with affinity for a desiredtarget molecule. The target molecule of the targeting moieties may, forexample, be a cell-surface antigen. Cell-surface antigens may bespecific for a cell or tissue type, such as, for example, cancer cells.Illustrative examples of such cell surface proteins are HER-2 orproteoglycans such as NEU-2.

As indicated above, a mutein of the invention may in some embodiments beconjugated to a moiety that extends the serum half-life of the mutein(in this regard see also PCT publication WO 2006/56464 where suchconjugation strategies are described with references to muteins of humanneutrophil gelatinase-associated lipocalin with binding affinity forCTLA-4). The moiety that extends the serum half-life may be apolyalkylene glycol molecule, hydroxyethyl starch, fatty acid molecules,such as palmitic acid (Vajo & Duckworth (2000) Pharmacol. Rev. 52, 1-9),an Fc part of an immunoglobulin, a CH3 domain of an immunoglobulin, aCH4 domain of an immunoglobulin, albumin or a fragment thereof, analbumin binding peptide, or an albumin binding protein, transferrin toname only a few. The albumin binding protein may be a bacterial albuminbinding protein, an antibody, an antibody fragment including domainantibodies (see U.S. Pat. No. 6,696,245, for example), or a lipocalinmutein with binding activity for albumin. Accordingly, suitableconjugation partners for extending the half-life of a lipocalin muteinof the invention include albumin (Osborn et al. (2002) J. Pharmacol.Exp. Ther. 303, 540-548), or an albumin binding protein, for example, abacterial albumin binding domain, such as the one of streptococcalprotein G (König, T. and Skerra, A. (1998) J. Immunol. Methods 218,73-83). Other examples of albumin binding peptides that can be used asconjugation partner are, for instance, those having aCys-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys consensus sequence, wherein Xaa₁ is Asp,Asn, Ser, Thr, or Trp; Xaa₂ is Asn, Gln, His, Ile, Leu, or Lys; Xaa₃ isAla, Asp, Phe, Trp, or Tyr; and Xaa₄ is Asp, Gly, Leu, Phe, Ser, or Thras described in US patent application 2003/0069395 or Dennis et al.(Dennis et al. (2002) J. Biol. Chem. 277, 35035-35043).

In other embodiments, albumin itself or a biological active fragment ofalbumin can be used as conjugation partner of a lipocalin mutein of theinvention. The term “albumin” comprises all mammal albumins such ashuman serum albumin or bovine serum albumin or rat albumin. The albuminor fragment thereof can be recombinantly produced as described in U.S.Pat. No. 5,728,553 or European patent applications EP 0 330 451 and EP 0361 991. Recombinant human albumin (Recombumin®) for use as a proteinstabilizer is for example available from Novozymes Delta Ltd.(Nottingham, UK).

If the albumin-binding protein is an antibody fragment it may be adomain antibody. Domain Antibodies (dAbs) are engineered to allowprecise control over biophysical properties and in vivo half-life tocreate the optimal safety and efficacy product profile. DomainAntibodies are for example commercially available from Domantis Ltd.(Cambridge, UK and MA, USA).

Using transferrin as a moiety to extend the serum half-life of themuteins of the invention, the muteins can be genetically fused to the Nor C terminus, or both, of non-glycosylated transferrin.Non-glycosylated transferrin has a half-life of 14-17 days, and atransferrin fusion protein will similarly have an extended half-life.The transferrin carrier also provides high bioavailability,biodistribution and circulating stability. This technology iscommercially available from BioRexis (BioRexis PharmaceuticalCorporation, PA, USA). Recombinant human transferrin (DeltaFerrin™) foruse as a protein stabilizer is also commercially available fromNovozymes Delta Ltd. (Nottingham, UK).

If an Fc part of an immunoglobulin is used for the purpose to prolongthe serum half-life of the muteins of the invention, the SynFusion™technology, commercially available from Syntonix Pharmaceuticals, Inc(MA, USA), may be used. The use of this Fc-fusion technology allows thecreation of longer-acting biopharmaceuticals and may for example consistof two copies of the mutein linked to the Fc region of an antibody toimprove pharmacokinetics, solubility, and production efficiency.

Yet another alternative to prolong the half-life of a mutein of theinvention is to fuse to the N- or C-terminus of a mutein of theinvention long, unstructured, flexible glycine-rich sequences (forexample poly-glycine with about 20 to 80 consecutive glycine residues).This approach disclosed in WO2007/038619, for example, has also beenterm “rPEG” (recombinant PEG).

If polyalkylene glycol is used as conjugation partner, the polyalkyleneglycol can be substituted or unsubstituted. It can also be an activatedpolyalkylene derivative. Examples of suitable compounds are polyethyleneglycol (PEG) molecules as described in WO 99/64016, in U.S. Pat. No.6,177,074 or in U.S. Pat. No. 6,403,564 in relation to interferon, or asdescribed for other proteins such as PEG-modified asparaginase,PEG-adenosine deaminase (PEG-ADA) or PEG-superoxide dismutase (see forexample, Fuertges et al. (1990) The Clinical Efficacy of Poly(EthyleneGlycol)-Modified Proteins J. Control. Release 11, 139-148). Themolecular weight of such a polymer, preferrably polyethylene glycol, mayrange from about 300 to about 70.000 Dalton, including, for example,polyethylene glycol with a molecular weight of about 10.000, of about20.000, of about 30.000 or of about 40.000 Dalton. Moreover, as e.g.described in U.S. Pat. No. 6,500,930 or 6,620,413, carbohydrate oligo-and polymers such as starch or hydroxyethyl starch (HES) can beconjugated to a mutein of the invention for the purpose of serumhalf-life extension.

If one of the above moieties is conjugated to the hNGAL mutein of theinvention, conjugation to an amino acid side chain can be advantageous.Suitable amino acid side chains may occur naturally in the amino acidsequence of hNGAL or may be introduced by mutagenesis. In case asuitable binding site is introduced via mutagenesis, one possibility isthe replacement of an amino acid at the appropriate position by acysteine residue. In one embodiment, such mutation includes theintroduction of a Cys residue at at least one of the sequence positionsthat correspond to sequence positions 14, 21, 60, 84, 88, 116, 141, 145,143, 146 or 158 of the wild type sequence of hNGAL. The newly createdcysteine residue at any of these positions can in the following beutilized to conjugate the mutein to moiety prolonging the serumhalf-life of the mutein, such as PEG or an activated derivative thereof.

In another embodiment, in order to provide suitable amino acid sidechains for conjugating one of the above moieties to the muteins of theinvention artificial amino acids may be introduced by mutagenesis.Generally, such artificial amino acids are designed to be more reactiveand thus to facilitate the conjugation to the desired moiety. Oneexample of such an artificial amino acid that may be introduced via anartificial tRNA is para-acetyl-phenylalanine.

For several applications of the muteins disclosed herein it may beadvantageous to use them in the form of fusion proteins. In someembodiments, the inventive hNGAL mutein is fused at its N-terminus orits C-terminus to a protein, a protein domain or a peptide such as asignal sequence and/or an affinity tag.

For pharmaceutical applications a mutein of the invention may be fusedto a fusion partner that extends the in vivo serum half-life of themutein (see again PCT publication WO 2006/56464 where suitable fusionpartner are described with references to muteins of human neutrophilegelatinase-associated lipocalin with binding affinity for CTLA-4).Similar to the conjugates described above, the fusion partner may be anFc part of an immunoglobulin, a CH3 domain of an immunoglobulin, a CH4domain of an immunogloubulin, albumin, an albumin binding peptide or analbumin binding protein, to name only a few. Again, the albumin bindingprotein may be a bacterial albumin binding protein or a lipocalin muteinwith binding activity for albumin. Accordingly, suitable fusion partnersfor extending the half-life of a lipocalin mutein of the inventioninclude albumin (Osborn, B. L. et al. (2002) supra J. Pharmacol. Exp.Ther. 303, 540-548), or an albumin binding protein, for example, abacterial albumin binding domain, such as the one of streptococcalprotein G (Konig, T. and Skerra, A. (1998) supra J. Immunol. Methods218, 73-83). The albumin binding peptides described in Dennis et al,supra (2002) or US patent application 2003/0069395 having aCys-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys consensus sequence, wherein Xaa₁ is Asp,Asn, Ser, Thr, or Trp; Xaa₂ is Asn, Gln, His, Ile, Leu, or Lys; Xaa₃ isAla, Asp, Phe, Tip, or Tyr; and Xaa₄ is Asp, Gly, Leu, Phe, Ser, or Thrcan also be used as fusion partner. It is also possible to use albuminitself or a biological active fragment of albumin as fusion partner of alipocalin mutein of the invention. The term “albumin” comprises allmammal albumins such as human serum albumin or bovine serum albumin orrat serum albumin. The recombinant production of albumin or fragmentsthereof is well known in the art and for example described in U.S. Pat.No. 5,728,553, European patent application EP 0 330 451 or EP 0 361 991.

The fusion partner may confer new characteristics to the inventivelipocalin mutein such as enzymatic activity or binding affinity forother molecules. Examples of suitable fusion proteins are alkalinephosphatase, horseradish peroxidase, gluthation-S-transferase, thealbumin-binding domain of protein G, protein A, antibody fragments,oligomerization domains, lipocalin muteins of same or different bindingspecificity (which results in the formation of “duocalins”, cf.Schlehuber, S., and Skerra, A. (2001), Duocalins, engineeredligand-binding proteins with dual specificity derived from the lipocalinfold. Biol. Chem. 382, 1335-1342), or toxins.

In particular, it may be possible to fuse a lipocalin mutein of theinvention with a separate enzyme active site such that both “components”of the resulting fusion protein together act on a given therapeutictarget. The binding domain of the lipocalin mutein attaches to thedisease-causing target, allowing the enzyme domain to abolish thebiological function of the target.

Affinity tags such as the Strep-tag® or Strep-tag® II (Schmidt, T. G. M.et al. (1996) J. Mol. Biol. 255, 753-766), the myc-tag, the FLAG-tag,the His₆-tag or the HA-tag or proteins such as glutathione-S-transferasealso allow easy detection and/or purification of recombinant proteinsare further examples of preferred fusion partners. Finally, proteinswith chromogenic or fluorescent properties such as the green fluorescentprotein (GFP) or the yellow fluorescent protein (YFP) are suitablefusion partners for a lipocalin mutein of the invention as well.

The term “fusion protein” as used herein also comprises lipocalinmuteins according to the invention containing a signal sequence. Signalsequences at the N-terminus of a polypeptide direct this polypeptide toa specific cellular compartment, for example the periplasm of E. coli orthe endoplasmatic reticulum of eukaryotic cells. A large number ofsignal sequences is known in the art. A preferred signal sequence forsecretion a polypeptide into the periplasm of E. coli is the OmpA-signalsequence.

The present invention also relates to nucleic acid molecules (DNA andRNA) comprising nucleotide sequences coding for muteins as describedherein. Since the degeneracy of the genetic code permits substitutionsof certain codons by other codons specifying the same amino acid, theinvention is not limited to a specific nucleic acid molecule encoding amutein of the invention but includes all nucleic acid moleculescomprising nucleotide sequences encoding a functional mutein.

Therefore, the present invention also includes a nucleic acid sequenceencoding a mutein according to the invention including a mutation at atleast one codon of any of the amino acid sequence positions 33, 36, 41,52, 54, 68, 70, 79, 81, 134, 136 and 138 of the linear polypeptidesequence of hNGAL.

The invention as disclosed herein also includes nucleic acid moleculesencoding hNGAL muteins, which comprise additional mutations outside theindicated sequence positions of experimental mutagenesis. Such mutationsare often tolerated or can even prove to be advantageous, for example ifthey contribute to an improved folding efficiency, serum stability,thermal stability or ligand binding affinity of the mutein.

A nucleic acid molecule disclosed in this application may be “operablylinked” to a regulatory sequence (or regulatory sequences) to allowexpression of this nucleic acid molecule.

A nucleic acid molecule, such as DNA, is referred to as “capable ofexpressing a nucleic acid molecule” or capable “to allow expression of anucleotide sequence” if it comprises sequence elements which containinformation regarding to transcriptional and/or translationalregulation, and such sequences are “operably linked” to the nucleotidesequence encoding the polypeptide. An operable linkage is a linkage inwhich the regulatory sequence elements and the sequence to be expressedare connected in a way that enables gene expression. The precise natureof the regulatory regions necessary for gene expression may vary amongspecies, but in general these regions comprise a promoter which, inprokaryotes, contains both the promoter per se, i.e. DNA elementsdirecting the initiation of transcription, as well as DNA elementswhich, when transcribed into RNA, will signal the initiation oftranslation. Such promoter regions normally include 5′ non-codingsequences involved in initiation of transcription and translation, suchas the −35/−10 boxes and the Shine-Dalgarno element in prokaryotes orthe TATA box, CAAT sequences, and 5′-capping elements in eukaryotes.These regions can also include enhancer or repressor elements as well astranslated signal and leader sequences for targeting the nativepolypeptide to a specific compartment of a host cell.

In addition, the 3′ non-coding sequences may contain regulatory elementsinvolved in transcriptional termination, polyadenylation or the like.If, however, these termination sequences are not satisfactory functionalin a particular host cell, then they may be substituted with signalsfunctional in that cell.

Therefore, a nucleic acid molecule of the invention can include aregulatory sequence, preferably a promoter sequence. In anotherpreferred embodiment, a nucleic acid molecule of the invention comprisesa promoter sequence and a transcriptional termination sequence. Suitableprokaryotic promoters are, for example, the tet promoter, the lacUV5promoter or the T7 promoter. Examples of promoters useful for expressionin eukaryotic cells are the SV40 promoter or the CMV promoter.

The nucleic acid molecules of the invention can also be part of a vectoror any other kind of cloning vehicle, such as a plasmid, a phagemid, aphage, a baculovirus, a cosmid or an artificial chromosome.

In one embodiment, the nucleic acid molecule is comprised in a phasmid.A phasmid vector denotes a vector encoding the intergenic region of atemperent phage, such as M13 or f1, or a functional part thereof fusedto the cDNA of interest. After superinfection of the bacterial hostcells with such an phagemid vector and an appropriate helper phage (e.g.M13K07, VCS-M13 or R408) intact phage particles are produced, therebyenabling physical coupling of the encoded heterologous cDNA to itscorresponding polypeptide displayed on the phage surface (reviewed,e.g., in Kay, B. K. et al. (1996) Phage Display of Peptides andProteins—A Laboratory Manual, 1st Ed., Academic Press, New York N.Y.;Lowman, H. B. (1997) Annu. Rev. Biophys. Biomol. Struct. 26, 401-424, orRodi, D. J., and Makowski, L. (1999) Curr. Opin. Biotechnol. 10, 87-93).

Such cloning vehicles can include, aside from the regulatory sequencesdescribed above and a nucleic acid sequence encoding a lipocalin muteinof the invention, replication and control sequences derived from aspecies compatible with the host cell that is used for expression aswell as selection markers conferring a selectable phenotype ontransformed or transfected cells. Large numbers of suitable cloningvectors are known in the art, and are commercially available.

The DNA molecule encoding lipocalin muteins of the invention, and inparticular a cloning vector containing the coding sequence of such alipocalin mutein can be transformed into a host cell capable ofexpressing the gene. Transformation can be performed using standardtechniques (Sambrook, J. et al. (1989), supra).

Thus, the invention is also directed to a host cell containing a nucleicacid molecule as disclosed herein.

The transformed host cells are cultured under conditions suitable forexpression of the nucleotide sequence encoding a fusion protein of theinvention. Suitable host cells can be prokaryotic, such as Escherichiacoli (E. coli) or Bacillus subtilis, or eukaryotic, such asSaccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells,immortalized mammalian cell lines (e.g. HeLa cells or CHO cells) orprimary mammalian cells

The invention also relates to a method for the production of a mutein ofthe invention, wherein the mutein, a fragment of the mutein or a fusionprotein of the mutein and another polypeptide is produced starting fromthe nucleic acid coding for the mutein by means of genetic engineeringmethods. The method can be carried out in vivo, the mutein can forexample be produced in a bacterial or eucaryotic host organism and thenisolated from this host organism or its culture. It is also possible toproduce a protein in vitro, for example by use of an in vitrotranslation system.

When producing the mutein in vivo a nucleic acid encoding a mutein ofthe invention is introduced into a suitable bacterial or eukaryotic hostorganism by means of recombinant DNA technology (as already outlinedabove). For this purpose, the host cell is first transformed with acloning vector comprising a nucleic acid molecule encoding a mutein ofthe invention using established standard methods (Sambrook, J. et al.(1989), supra). The host cell is then cultured under conditions, whichallow expression of the heterologous DNA and thus the synthesis of thecorresponding polypeptide. Subsequently, the polypeptide is recoveredeither from the cell or from the cultivation medium.

In one aspect, the present invention relates to a method for thegeneration of a mutein of the invention, comprising:

-   -   (a) subjecting a nucleic acid molecule encoding an hNGAL protein        to mutagenesis at a nucleotide triplet coding for at least one        of any of the sequence positions corresponding to the sequence        positions 33, 36, 41, 52, 54, 68, 70, 79, 81, 134, 136, and 138        of the linear polypeptide sequence of hNGAL, resulting in one or        more mutein nucleic acid molecule(s)    -   (b) expressing the one more mutein nucleic acid molecule(s)        obtained in (a) in a suitable expression system, and    -   (c) enriching the one or more mutein(s) having a detectable        binding affinity for a given target by means of selection and/or        isolation.

The term “mutagenesis” as used herein means that the experimentalconditions are chosen such that the amino acid naturally occurring at agiven sequence position of hNGAL (Swiss-Prot data bank entry P80188) canbe substituted by at least one amino acid that is not present at thisspecific position in the respective natural polypeptide sequence. Theterm “mutagenesis” also includes the (additional) modification of thelength of sequence segments by deletion or insertion of one or moreamino acids. Thus, it is within the scope of the invention that, forexample, one amino acid at a chosen sequence position is replaced by astretch of three random mutations, leading to an insertion of two aminoacid residues compared to the length of the respective segment of thewild type protein. Such an insertion of deletion may be introducedindependently from each other in any of the peptide segments that can besubjected to mutagenesis in the invention. In one exemplary embodimentof the invention, an insertion of several mutations may be introducedinto the loop AB of the chosen lipocalin scaffold (cf. InternationalPatent Application WO 2005/019256 which is incorporated by reference itsentirety herein). The term “random mutagenesis” means that nopredetermined single amino acid (mutation) is present at a certainsequence position but that at least two amino acids can be incorporatedwith a certain probability at a predefined sequence position duringmutagenesis.

The coding sequence of hNGAL is used as a starting point for themutagenesis of the peptide segments selected in the present invention.For the mutagenesis of the recited amino acid positions, the personskilled in the art has at his disposal the various established standardmethods for site-directed mutagenesis (Sambrook, J. et al. (1989),supra). A commonly used technique is the introduction of mutations bymeans of PCR (polymerase chain reaction) using mixtures of syntheticoligonucleotides, which bear a degenerate base composition at thedesired sequence positions. For example, use of the codon NNK or NNS(wherein N=adenine, guanine or cytosine or thymine; K=guanine orthymine; S=adenine or cytosine) allows incorporation of all 20 aminoacids plus the amber stop codon during mutagenesis, whereas the codonVVS limits the number of possibly incorporated amino acids to 12, sinceit excludes the amino acids Cys, Ile, Leu, Met, Phe, Trp, Tyr, Val frombeing incorporated into the selected position of the polypeptidesequence; use of the codon NMS (wherein M=adenine or cytosine), forexample, restricts the number of possible amino acids to 11 at aselected sequence position since it excludes the amino acids Arg, Cys,Gly, Ile, Leu, Met, Phe, Trp, Val from being incorporated at a selectedsequence position. In this respect it is noted that codons for otheramino acids (than the regular 20 naturally occurring amino acids) suchas selenocystein or pyrrolysine can also be incorporated into a nucleicacid of a mutein. It is also possible, as described by Wang, L., et al.(2001) Science 292, 498-500, or Wang, L., and Schultz, P. G. (2002)Chem. Comm. 1, 1-11, to use “artificial” codons such as UAG which areusually recognized as stop codons in order to insert other unusual aminoacids, for example o-methyl-L-tyrosine or p-aminophenylalanine.

The use of nucleotide building blocks with reduced base pairspecificity, as for example inosine, 8-oxo-2′deoxyguanosine or6(2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimindo-1,2-oxazine-7-one(Zaccolo et al. (1996) J. Mol. Biol. 255, 589-603), is another optionfor the introduction of mutations into a chosen sequence segment.

A further possibility is the so-called triplet-mutagenesis. This methoduses mixtures of different nucleotide triplets, each of which codes forone amino acid, for incorporation into the coding sequence (Virnekäs B,Ge L, Plückthun A, Schneider K C, Wellnhofer G, Moroney S E. 1994Trinucleotide phosphoramidites: ideal reagents for the synthesis ofmixed oligonucleotides for random mutagenesis. Nucleic Acids Res 22,5600-5607).

One possible strategy for introducing mutations in the selected regionsof the respective polypeptides is based on the use of fouroligonucleotides, each of which is partially derived from one of thecorresponding sequence segments to be mutated. When synthesizing theseoligonucleotides, a person skilled in the art can employ mixtures ofnucleic acid building blocks for the synthesis of those nucleotidetriplets which correspond to the amino acid positions to be mutated sothat codons encoding all natural amino acids randomly arise, which atlast results in the generation of a lipocalin peptide library. Forexample, the first oligonucleotide corresponds in its sequence—apartfrom the mutated positions—to the coding strand for the peptide segmentto be mutated at the most N-terminal position of the lipocalinpolypeptide. Accordingly, the second oligonucleotide corresponds to thenon-coding strand for the second sequence segment following in thepolypeptide sequence. The third oligonucleotide corresponds in turn tothe coding strand for the corresponding third sequence segment. Finally,the fourth oligonucleotide corresponds to the non-coding strand for thefourth sequence segment. A polymerase chain reaction can be performedwith the respective first and second oligonucleotide and separately, ifnecessary, with the respective third and fourth oligonucleotide.

The amplification products of both of these reactions can be combined byvarious known methods into a single nucleic acid comprising the sequencefrom the first to the fourth sequence segments, in which mutations havebeen introduced at the selected positions. To this end, both of theproducts can for example be subjected to a new polymerase chain reactionusing flanking oligonucleotides as well as one or more mediator nucleicacid molecules, which contribute the sequence between the second and thethird sequence segment. In the choice of the number and arrangementwithin the sequence of the oligonucleotides used for the mutagenesis,the person skilled in the art has numerous alternatives at his disposal.

The nucleic acid molecules defined above can be connected by ligationwith the missing 5′- and 3′-sequences of a nucleic acid encoding alipocalin polypeptide and/or the vector, and can be cloned in a knownhost organism. A multitude of established procedures are available forligation and cloning (Sambrook, J. et al. (1989), supra). For example,recognition sequences for restriction endonucleases also present in thesequence of the cloning vector can be engineered into the sequence ofthe synthetic oligonucleotides. Thus, after amplification of therespective PCR product and enzymatic cleavage the resulting fragment canbe easily cloned using the corresponding recognition sequences.

Longer sequence segments within the gene coding for the protein selectedfor mutagenesis can also be subjected to random mutagenesis via knownmethods, for example by use of the polymerase chain reaction underconditions of increased error rate, by chemical mutagenesis or by usingbacterial mutator strains. Such methods can also be used for furtheroptimization of the target affinity or specificity of a lipocalinmutein. Mutations possibly occurring outside the segments ofexperimental mutagenesis are often tolerated or can even prove to beadvantageous, for example if they contribute to an improved foldingefficiency or folding stability of the lipocalin mutein.

According to one embodiment of the present invention, the above methodincludes subjecting the nucleic acid molecule encoding an hNGAL proteinto mutagenesis at at least 9, 10, 11 or all 12 nucleotide tripletscoding for any of the above indicated sequence positions of hNGAL.

In one further embodiment, the method further includes subjecting thenucleic acid molecule to mutagenesis at at least one nucleotide tripletcoding for any of the sequence positions corresponding to the sequencepositions 42, 48, 49, 55, 75, 77, 80, and 127 of the linear polypeptidesequence of hNGAL. The method may further include subjecting the nucleicacid molecule to mutagenesis at at least one nucleotide triplet codingfor any of the sequence positions corresponding to the sequencepositions 43, 44, 46, 47, 50, 51, 59, 65, 78, 86, 87, 98, 99, 103, 107,110 and 111 of the linear polypeptide sequence of hNGAL.

In still another embodiment of the present invention, the method furtherincludes subjecting the nucleic acid molecule to mutagenesis atnucleotide triplets coding for at least any 9 of the sequence positionscorresponding to the sequence positions 33, 36, 41, 42, 48, 49, 52, 54,55, 68, 70, 75, 77, 79, 80, 81, 127, 134, 136 and 138 of the linearpolypeptide sequence of hNGAL.

In a still further embodiment, the method includes subjecting thenucleic acid molecule to mutagenesis at nucleotide triplets coding forat least any 9 of the sequence positions corresponding to the sequencepositions 33, 36, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 54, 55,59, 65, 68, 70, 75, 77, 78, 79, 80, 81, 86, 87, 98, 99, 103, 107, 110,111, 127, 134, 136 and 138 of the linear polypeptide sequence of hNGAL.

According to the method of the invention a mutein is obtained startingfrom a nucleic acid encoding hNGAL. Such a nucleic acid is subjected tomutagenesis and introduced into a suitable bacterial or eukaryotic hostorganism by means of recombinant DNA technology. Obtaining a nucleicacid library of hNGAL can be carried out using any suitable techniquethat is known in the art for generating lipocalin muteins withantibody-like properties, i.e. muteins that have affinity towards agiven target. Examples of such combinatorial methods are described indetail in the international patent applications WO 99/16873, WO00/75308, WO 03/029471, WO 03/029462, WO 03/029463, WO 2005/019254, WO2005/019255, WO 2005/019256, or WO 2006/56464 for instance. The contentof each of these patent applications is incorporated by reference hereinin its entirety. After expression of the nucleic acid sequences thatwere subjected to mutagenesis in an appropriate host, the clonescarrying the genetic information for the plurality of respectivelipocalin muteins, which bind a given target can be selected from thelibrary obtained. Well known techniques can be employed for theselection of these clones, such as phage display (reviewed in Kay, B. K.et al. (1996) supra; Lowman, H. B. (1997) supra or Rodi, D. J., andMakowski, L. (1999) supra), colony screening (reviewed in Pini, A. etal. (2002) Comb. Chem. High Throughput Screen. 5, 503-510), ribosomedisplay (reviewed in Amstutz, P. et al. (2001) Curr. Opin. Biotechnol.12, 400-405) or mRNA display as reported in Wilson, D. S. et al. (2001)Proc. Natl. Acad. Sci. USA 98, 3750-3755 or the methods specificallydescribed in WO 99/16873, WO 00/75308, WO 03/029471, WO 03/029462, WO03/029463, WO 2005/019254, WO 2005/019255, WO 2005/019256, or WO2006/56464.

In accordance with this disclosure, step (c) further comprises inanother embodiment of the above methods:

-   -   (i) providing as a given ligand a compound selected from the        group consisting of a chemical compound in free or conjugated        form that exhibits features of an immunological hapten, a        peptide, a protein or another macromolecule such as a        polysaccharide, a nucleic acid molecule (DNA or RNA, for        example) or an entire virus particle or viroid, for example,    -   (ii) contacting the plurality of muteins with said ligand in        order to allow formation of complexes between said ligand and        muteins having binding affinity for said ligand, and    -   (iii) removing muteins having no or no substantial binding        affinity.

In specific embodiments of the invention, the ligand may be a smallorganic molecule, such as a metal-chelating agent.

In one embodiment of the methods of the invention, the selection in step(c) is carried out under competitive conditions. Competitive conditionsas used herein means that selection of muteins encompasses at least onestep in which the muteins and the given non-natural ligand of hNGAL(target) are brought in contact in the presence of an additional ligand,which competes with binding of the muteins to the target. Thisadditional ligand may be a physiological ligand of the target, an excessof the target itself or any other non-physiological ligand of the targetthat binds at least an overlapping epitope to the epitope recognized bythe muteins of the invention and thus interferes with target binding ofthe muteins. Alternatively, the additional ligand competes with bindingof the muteins by complexing an epitope distinct from the binding siteof the muteins to the target by allosteric effects.

An embodiment of the phage display technique (reviewed in Kay, B. K. etal. (1996), supra; Lowman, H. B. (1997) supra or Rodi, D. J., andMakowski, L. (1999), supra) using temperent M13 phage is given as anexample of a selection method that can be employed in the presentinvention. Another embodiment of the phage display technology that canbe used for selection of muteins of the invention is the hyperphagephage technology as described by Broders et al. (Broders et al. (2003)“Hyperphage. Improving antibody presentation in phage display.” MethodsMol. Biol. 205:295-302). Other temperent phage such as f1 or lytic phagesuch as T7 may be employed as well. For the exemplary selection method,M13 phagemids are produced which allow the expression of the mutatedlipocalin nucleic acid sequence as a fusion protein with a signalsequence at the N-terminus, preferably the OmpA-signal sequence, andwith the capsid protein pIII of the phage M13 or fragments thereofcapable of being incorporated into the phage capsid at the C-terminus.The C-terminal fragment ApIII of the phage capsid protein comprisingamino acids 217 to 406 of the wild type sequence is preferably used toproduce the fusion proteins. Especially preferred in one embodiment is aC-terminal fragment of pIII, in which the cysteine residue at position201 is missing or is replaced by another amino acid.

Accordingly, a further embodiment of the methods of the inventioninvolves operably fusing a nucleic acid coding for the plurality ofmuteins of hNGAL and resulting from mutagenesis at the 3′ end with agene coding for the coat protein pIII of a filamentous bacteriophage ofthe M13-family or for a fragment of this coat protein, in order toselect at least one mutein for the binding of a given ligand.

The fusion protein may comprise additional components such as anaffinity tag, which allows the immobilization, detection and/orpurification of the fusion protein or its parts. Furthermore, a stopcodon can be located between the sequence regions encoding the lipocalinor its muteins and the phage capsid gene or fragments thereof, whereinthe stop codon, preferably an amber stop codon, is at least partiallytranslated into an amino acid during translation in a suitablesuppressor strain.

For example, the phasmid vector pTLPC27, now also called pTlc27 that isdescribed here can be used for the preparation of a phagemid libraryencoding hNGAL muteins. The inventive nucleic acid molecules coding forthe hNGAL muteins are inserted into the vector using the two BstXIrestriction sites. After ligation a suitable host strain such as E. coliXL1-Blue is transformed with the resulting nucleic acid mixture to yielda large number of independent clones. A respective vector can begenerated for the preparation of a hyperphagemid library, if desired.

The resulting library is subsequently superinfected in liquid culturewith an appropriate M13-helper phage or hyperphage in order to producefunctional phagemids. The recombinant phagemid displays the lipocalinmutein on its surface as a fusion with the coat protein pIII or afragment thereof, while the N-terminal signal sequence of the fusionprotein is normally cleaved off. On the other hand, it also bears one ormore copies of the native capsid protein pIII supplied by the helperphage and is thus capable of infecting a recipient, in general abacterial strain carrying an F- or F′-plasmid. In case of hyperphagedisplay, the hyperphagemids display the lipocalin muteins on theirsurface as a fusion with the infective coat protein pIII but no nativecapsid protein. During or after infection with helper phage orhyperphage, gene expression of the fusion protein between the lipocalinmutein and the capsid protein pIII can be induced, for example byaddition of anhydrotetracycline. The induction conditions are chosensuch that a substantial fraction of the phagemids obtained displays atleast one lipocalin mutein on their surface. In case of hyperphagedisplay induction conditions result in a population of hyperphagemidscarrying between three and five fusion proteins consisting of thelipocalin mutein and the capsid protein pIII. Various methods are knownfor isolating the phagemids, such as precipitation with polyethyleneglycol. Isolation typically occurs after an incubation period of 6-8hours.

The isolated phasmids can then be subjected to selection by incubationwith the desired target, wherein the target is presented in a formallowing at least temporary immobilization of those phagemids whichcarry muteins with the desired binding activity as fusion proteins intheir coat. Among the various embodiments known to the person skilled inthe art, the target can, for example, be conjugated with a carrierprotein such as serum albumin and be bound via this carrier protein to aprotein binding surface, for example polystyrene. Microtiter platessuitable for ELISA techniques or so-called “immuno-sticks” canpreferrably be used for such an immobilization of the target.Alternatively, conjugates of the target with other binding groups, suchas biotin, can be used. The target can then be immobilized on a surfacewhich selectively binds this group, for example microtiter plates orparamagnetic particles coated with streptavidin, neutravidin or avidin.If the target is fused to an Fc portion of an immunoglobulin,immobilization can also be achieved with surfaces, for examplemicrotiter plates or paramagnetic particles, which are coated withprotein A or protein G.

Non-specific phagemid-binding sites present on the surfaces can besaturated with blocking solutions as they are known for ELISA methods.The phagemids are then typically brought into contact with the targetimmobilized on the surface in the presence of a physiological buffer.Unbound phagemids are removed by multiple washings. The phagemidparticles remaining on the surface are then eluted. For elution, severalmethods are possible. For example, the phagemids can be eluted byaddition of proteases or in the presence of acids, bases, detergents orchaotropic salts or under moderately denaturing conditions. A preferredmethod is the elution using buffers of pH 2.2, wherein the eluate issubsequently neutralized. Alternatively, a solution of the free targetcan be added in order to compete with the immobilized target for bindingto the phagemids or target-specific phagemids can be eluted bycompetition with immunoglobulins or natural liganding proteins whichspecifically bind to the target of interest.

Afterwards, E. coli cells are infected with the eluted phagemids.Alternatively, the nucleic acids can be extracted from the elutedphagemids and used for sequence analysis, amplification ortransformation of cells in another manner. Starting from the E. coliclones obtained in this way, fresh phagemids or hyperphagemids are againproduced by superinfection with M13 helper phages or hyperphageaccording to the method described above and the phagemids amplified inthis way are once again subjected to a selection on the immobilizedtarget. Multiple selection cycles are often necessary in order to obtainthe phagemids with the muteins of the invention in sufficiently enrichedform. The number of selection cycles is preferably chosen such that inthe subsequent functional analysis at least 0.1% of the clones studiedproduce muteins with detectable affinity for the given target. Dependingon the size, i.e. the complexity of the library employed, 2 to 8 cyclesare typically required to this end.

For the functional analysis of the selected muteins, an E. coli strainis infected with the phagemids obtained from the selection cycles andthe corresponding double stranded phasmid DNA is isolated. Starting fromthis phasmid DNA, or also from the single-stranded DNA extracted fromthe phagemids, the nucleic acid sequences of the selected muteins of theinvention can be determined by the methods known in the art and theamino acid sequence can be deduced therefrom. The mutated region or thesequence of the entire hNGAL mutein can be subcloned on anotherexpression vector and expressed in a suitable host organism. Forexample, the vector pTLPC26 now also called pTlc26 can be used forexpression in E. coli strains such as E. coli TG1. The muteins of hNGALthus produced can be purified by various biochemical methods. The hNGALmuteins produced, for example with pTlc26, carry the affinity peptideStrep-tag II (Schmidt et al., supra) at their C-termini and cantherefore preferably be purified by streptavidin affinitychromatography.

The selection can also be carried out by means of other methods. Manycorresponding embodiments are known to the person skilled in the art orare described in the literature. Moreover, a combination of methods canbe applied. For example, clones selected or at least enriched by “phagedisplay” can additionally be subjected to “colony screening”. Thisprocedure has the advantage that individual clones can directly beisolated with respect to the production of an hNGAL mutein withdetectable binding affinity for a target.

In addition to the use of E. coli as host organism in the “phagedisplay” technique or the “colony screening” method, other bacterialstrains, yeast or also insect cells or mammalian cells can be used forthis purpose. Further to the selection of an hNGAL mutein from a randomlibrary as described above, evolutive methods including limitedmutagenesis can also be applied in order to optimize a mutein thatalready possesses some binding activity for the target with respect toaffinity or specificity for the target after repeated.

Once a mutein with affinity to a given target has been selected, it isadditionally possible to subject such a mutein to another mutagenesis inorder to subsequently select variants of even higher affinity orvariants with improved properties such as higher thermostability,improved serum stability, thermodynamic stability, improved solubility,improved monomeric behavior, improved resistance against thermaldenaturation, chemical denaturation, proteolysis, or detergents etc.This further mutagenesis, which in case of aiming at higher affinity canbe considered as in vitro “affinity maturation”, can be achieved by sitespecific mutation based on rational design or a random mutation. Anotherpossible approach for obtaining a higher affinity or improved propertiesis the use of error-prone PCR, which results in point mutations over aselected range of sequence positions of the lipocalin mutein. Theerror-prone PCR can be carried out in accordance with any known protocolsuch as the one described by Zaccolo et al. (1996) J. Mol. Biol. 255,589-603. Other methods of random mutagenesis thatare suitable for suchpurposes include random insertion/deletion (RID) mutagenesis asdescribed by Murakami et al. (2002) Nat. Biotechnol. 20, 76-81 ornonhomologous random recombination (NRR) as described by Bittker et al.(2002) Nat. Biotechnol. 20, 1024-1029. If desired, affinity maturationcan also be carried out according to the procedure described in WO00/75308 or Schlehuber et al. (2000) J. Mol. Biol. 297, 1105-1120, wheremuteins of the bilin-binding protein having high affinity to digoxigeninwere obtained. A further approach for improving the affinity is to carryout positional saturation mutagenesis. In this approach “small” nucleicacid libraries can be created in which amino acid exchanges/mutationsare only introduced at single positions within any of the four loopsegments. These libraries are then directly subjected to a selectionstep (affinity screening) without further rounds of panning. Thisapproach allows the identification of residues that contribute toimproved binding of the desired target and allows identification of “hotspots” that are important for the binding.

In one embodiment, the above method for modifying a mutein furtherincludes introducing a Cys residue at at least one of any of thesequence positions that correspond to sequence positions 14, 21, 60, 84,88, 116, 141, 145, 143, 146 or 158 of the wild type sequence of hNGALand coupling a moiety that is able to modify the serum half time of saidmutein via the thiol group of a Cys residue introduced at at least oneof any of the sequence positions that correspond to sequence positions14, 21, 60, 84, 88, 116, 141, 145, 143, 146 or 158 of the wild typesequence of hNGAL. The moiety that is able to modify the serum half timeof said mutein may be selected from the group consisting of apolyalkylene glycol molecule and hydroxyethyl starch.

In a further aspect, the present invention is directed to a mutein ofhNGAL having detectable binding affinity to a given non-natural ligandof hNGAL, which is obtainable by or obtained by the above-detailedmethods of the invention.

In some hNGAL muteins of the invention, the naturally occurringdisulfide bond between Cys 76 and Cys 175 is removed. Accordingly, suchmuteins (or any other hNGAL mutein that does not comprise anintramolecular disulfide bond) can be produced in a cell compartmenthaving a reducing redox milieu, for example, in the cytoplasma ofGram-negative bacteria.

In case a lipocalin mutein of the invention comprises intramoleculardisulfide bonds, it may be preferred to direct the nascent polypeptideto a cell compartment having an oxidizing redox milieu using anappropriate signal sequence. Such an oxidizing environment may beprovided by the periplasm of Gram-negative bacteria such as E. coli, inthe extracellular milieu of Gram-positive bacteria or in the lumen ofthe endoplasmatic reticulum of eukaryotic cells and usually favors theformation of structural disulfide bonds.

It is, however, also possible to produce a mutein of the invention inthe cytosol of a host cell, preferably E. coli. In this case, thepolypeptide can either be directly obtained in a soluble and foldedstate or recovered in form of inclusion bodies, followed by renaturationin vitro. A further option is the use of specific host strains having anoxidizing intracellular milieu, which may thus allow the formation ofdisulfide bonds in the cytosol (Venturi et al. (2002) J. Mol. Biol. 315,1-8).

However, a mutein of the invention may not necessarily be generated orproduced only by use of genetic engineering. Rather, a lipocalin muteincan also be obtained by chemical synthesis such as Merrifield solidphase polypeptide synthesis or by in vitro transcription andtranslation. It is for example possible that promising mutations areidentified using molecular modeling and then to synthesize the wanted(designed) polypeptide in vitro and investigate the binding activity fora given target. Methods for the solid phase and/or solution phasesynthesis of proteins are well known in the art (reviewed, e.g., inLloyd-Williams et al. (1997) Chemical Approaches to the Synthesis ofPeptides and Proteins. CRC Press, Boca Raton, Fields, G B, and Colowick(1997) Solid-Phase Peptide Synthesis. Academic Press, San Diego, orBruckdorfer et al. (2004) Curr. Pharm. Biotechnol. 5, 29-43).

In another embodiment, the muteins of the invention may be produced byin vitro transcription/translation employing well-established methodsknown to those skilled in the art.

The invention also relates to a pharmaceutical composition comprising atleast one inventive mutein of hNGAL or a fusion protein or conjugatethereof and, optionally, a pharmaceutically acceptable excipient.

The lipocalin muteins according to the invention can be administered viaany parenteral or non-parenteral (enteral) route that is therapeuticallyeffective for proteinaceous drugs. Parenteral application methodscomprise, for example, intracutaneous, subcutaneous, intramuscular orintravenous injection and infusion techniques, e.g. in the form ofinjection solutions, infusion solutions or tinctures, as well as aerosolinstallation and inhalation, e.g. in the form of aerosol mixtures,sprays or powders. Non-parenteral delivery modes are, for instance,orally, e.g. in the form of pills, tablets, capsules, solutions orsuspensions, or rectally, e.g. in the form of suppositories. The muteinsof the invention can be administered systemically or topically informulations containing conventional non-toxic pharmaceuticallyacceptable excipients or carriers, additives and vehicles as desired.

In one embodiment of the present invention the pharmaceutical isadministered parenterally to a mammal, and in particular to humans.Corresponding administration methods include, but are not limited to,for example, intracutaneous, subcutaneous, intramuscular or intravenousinjection and infusion techniques, e.g. in the form of injectionsolutions, infusion solutions or tinctures as well as aerosolinstallation and inhalation, e.g. in the form of aerosol mixtures,sprays or powders. A combination of intravenous and subcutaneousinfusion and/or injection might be most convenient in case of compoundswith a relatively short serum half life. The pharmaceutical compositionmay be an aqueous solution, an oil-in water emulsion or a water-in-oilemulsion.

In this regard it is noted that transdermal delivery technologies, e.g.iontophoresis, sonophoresis or microneedle-enhanced delivery, asdescribed in Meidan and Michniak (2004) Am. J. Ther. 11(4), 312-316, canalso be used for transdermal delivery of the muteins described herein.Non-parenteral delivery modes are, for instance, oral, e.g. in the formof pills, tablets, capsules, solutions or suspensions, or rectaladministration, e.g. in the form of suppositories. The muteins of theinvention can be administered systemically or topically in formulationscontaining a variety of conventional non-toxic pharmaceuticallyacceptable excipients or carriers, additives, and vehicles.

The dosage of the mutein applied may vary within wide limits to achievethe desired preventive effect or therapeutic response. It will, forinstance, depend on the affinity of the compound for a chosen ligand aswell as on the half-life of the complex between the mutein and theligand in vivo. Further, the optimal dosage will depend on thebiodistribution of the mutein or its fusion protein or its conjugate,the mode of administration, the severity of the disease/disorder beingtreated as well as the medical condition of the patient. For example,when used in an ointment for topical applications, a high concentrationof the hNGAL mutein can be used. However, if wanted, the mutein may alsobe given in a sustained release formulation, for example liposomaldispersions or hydrogel-based polymer microspheres, like PolyActive™ orOctoDEX™ (cf. Bos et al., Business Briefing: Pharmatech 2003: 1-6).

Accordingly, the muteins of the present invention can be formulated intocompositions using pharmaceutically acceptable ingredients as well asestablished methods of preparation (Gennaro and Gennaro (2000)Remington: The Science and Practice of Pharmacy, 20th Ed., LippincottWilliams & Wilkins, Philadelphia, Pa.). To prepare the pharmaceuticalcompositions, pharmaceutically inert inorganic or organic excipients canbe used. To prepare e.g. pills, powders, gelatine capsules orsuppositories, for example, lactose, talc, stearic acid and its salts,fats, waxes, solid or liquid polyols, natural and hardened oils can beused. Suitable excipients for the production of solutions, suspensions,emulsions, aerosol mixtures or powders for reconstitution into solutionsor aerosol mixtures prior to use include water, alcohols, glycerol,polyols, and suitable mixtures thereof as well as vegetable oils.

The pharmaceutical composition may also contain additives, such as, forexample, fillers, binders, wetting agents, glidants, stabilizers,preservatives, emulsifiers, and furthermore solvents or solubilizers oragents for achieving a depot effect. The latter is that fusion proteinsmay be incorporated into slow or sustained release or targeted deliverysystems, such as liposomes and microcapsules.

The formulations can be sterilized by numerous means, includingfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile medium justprior to use.

A mutein of the present invention or a fusion protein or a conjugatethereof can be employed in many applications. In general, such a muteincan be used in all applications antibodies are used, except those withspecifically rely on the glycosylation of the Fc part.

Therefore, in another aspect of the invention, the invented muteins ofhNGAL are used for the binding and/or detection of a given non-naturalligand of hNGAL. Such use may comprise the steps of contacting themutein with a sample suspected of containing the given ligand undersuitable conditions, thereby allowing formation of a complex between themutein and the given ligand, and detecting the complexed mutein by asuitable signal.

The detectable signal can be caused by a label, as explained above, orby a change of physical properties due to the binding, i.e. the complexformation, itself. One example is plasmon surface resonance, the valueof which is changed during binding of binding partners from which one isimmobilized on a surface such as a gold foil.

The muteins of hNGAL disclosed herein may also be used for theseparation of a given non-natural ligand of hNGAL. Such use may comprisethe steps of contacting the mutein with a sample supposed to containsaid ligand under suitable conditions, thereby allowing formation of acomplex between the mutein and the given ligand, and separating themutein/ligand complex from the sample.

In both the use of the mutein for the detection of a given non-naturalligand as well as the separation of a given ligand, the mutein and/orthe target may be immobilized on a suitable solid phase.

The hNGAL muteins of the invention may also be used to target a compoundto a pre-selected site. In one such embodiment, a mutein of hNGAL isused for the targeting of a pharmaceutically active compound to apre-selected site in an organism or tissue, comprising of:

-   -   a) conjugating the mutein with said compound, and    -   b) delivering the mutein/compound complex to the pre-selected        site.

For such a purpose the mutein is contacted with the compound of interestin order to allow complex formation. Then the complex comprising themutein and the compound of interest are delivered to the pre-selectedsite. This may, for example, be achieved by coupling the mutein to atargeting moiety, such as an antibody, antibody fragment or lipocalinmutein or lipocalin mutein fragment with binding affinity for theselected target.

This use is in particular suitable, but not restricted to, fordelivering a drug (selectively) to a pre-selected site in an organism,such as an infected body part, tissue or organ which is supposed to betreated with the drug. Besides formation of a complex between mutein andcompound of interest, the mutein can also be reacted with the givencompound to yield a conjugate of mutein and compound. Similar to theabove complex, such a conjugate may be suitable to deliver the compoundto the pre-selected target site. Such a conjugate of mutein and compoundmay also include a linker that covalently links mutein and compound toeach other. Optionally, such a linker is stable in the bloodstream butis cleavable in a cellular environment.

The muteins disclosed herein and its derivatives can thus be used inmany fields similar to antibodies or fragments thereof. In addition totheir use for binding to a support, allowing the target of a givenmutein or a conjugate or a fusion protein of this target to beimmobilized or separated, the muteins can be used for labeling with anenzyme, an antibody, a radioactive substance or any other group havingbiochemical activity or defined binding characteristics. By doing so,their respective targets or conjugates or fusion proteins thereof can bedetected or brought in contact with them. For example, muteins of theinvention can serve to detect chemical structures by means ofestablished analytical methods (e.g. ELISA or Western Blot) or bymicroscopy or immunosensorics. Here, the detection signal can either begenerated directly by use of a suitable mutein conjugate or fusionprotein or indirectly by immunochemical detection of the bound muteinvia an antibody.

Numerous possible applications for the inventive muteins also exist inmedicine. In addition to their use in diagnostics and drug delivery, amutant polypeptide of the invention, which binds, for example, tissue-or tumor-specific cellular surface molecules can be generated. Such amutein may, for example, be employed in conjugated form or as a fusionprotein for “tumor imaging” or directly for cancer therapy.

Thus, the present invention also involves the use of the hNGAL muteinsof the invention for complex formation with a given non-natural ligandor target.

In a further aspect, the present invention also encompasses the use of amutein according to the invention for the manufacture of apharmaceutical composition. The pharmaceutical composition thus obtainedmay be suited for use in radio-immuno therapy (RIT) or for in vivoimaging. The pharmaceutical composition may be used as monotherapy or ascombination therapy.

In still another aspect, the present invention features a diagnostic oranalytical kit comprising a mutein according to the present invention.

Another aspect of the present invention relates to a method of treatinga subject with radioimmunotherapy (RIT), including administering amutein of the invention or a pharmaceutical composition comprising amutein of the invention to a subject in need thereof. The subject may beafflicted by a disease or disorder amenable to such treatment, and maybe, for example, cancer or another cell-proliferative disorder.

The subject in need of such a treatment may be a mammal, such as ahuman, a dog, a mouse, a rat, a pig, an ape such as cymologous monkeysto name only a few illustrative examples.

In still another aspect, the present invention features a method for invivo imaging in a subject, including administering to said subject amutein of the invention or a pharmaceutical composition comprising amutein of the invention. The subject may be defined as above.

The invention is further illustrated by the following non-limitingExamples and the attached drawings in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B show the three-dimensional structure of hNGAL in complex withenterobactin together with a schematic representation of the designedrandom library of hNGAL.

FIGS. 2A-2E show the properties of hNGAL variants with Me•DTPA bindingactivity.

FIG. 3 shows the crystal structure of hNGAL variants with Me•DTPAbinding activity.

FIG. 4 shows a potential application of hNGAL variants with Me•DTPAbinding activity for pretargeting radioimmunotherapy or in vivo imaging.

FIG. 1A shows the three-dimensional structure of human hNGAL in complexwith enterobactin (PDB entry 1L6M, chain A, containing the intactligand; courtesy of Dr. Roland Strong). The polypeptide backbone isshown as shown as ribbon in light grey whereas the natural ligand iscolored black. Side chains randomized in the initial “naive” library areshown in grey.

FIG. 1B shows a schematic representation of the assembly PCR strategyfor the simultaneous random mutagenesis of the 12 amino acid positions33, 36, 41, 52, 54, 68, 70, 79, 81, 134, 136, and 138. The structuralgene of NGAL was used as template in a PCR with the degenerateoligodeoxynucleotides P1 and P2, resulting in fragment (a), and with thedegenerate primers P3 and P4, resulting in fragment (b). Randomizedpositions are indicated by light bars. Both fragments were isolated,combined and applied in a next amplification carried out with PCRprimers P5 and P6. Two different BstXI restriction sites were used forsubcloning of the gene cassette on pNGAL35, a plasmid vector for phagedisplay.

FIGS. 2A-2E show the properties of hNGAL variants with Me•DTPA bindingactivity.

FIG. 2A shows SDS-PAGE analysis of recombinant wild type hNGAL (lanes1,4) and variants Tb7.N9 (lanes 2,5) as well as C26 (lanes 3,6) afterStrep-tag II affinity purification and gel filtration. Lanes 1-3 showsamples reduced with 2-mercaptoethanol. The slightly enhancedelectrophoretic mobility under non-reducing conditions indicates properformation of the single disulphide bond in each case.

FIG. 2B depicts binding activity in the ELISA. A microtiter plate wascoated with the purified hNGAL variants, captured via an antibodyspecific for the Strep-tag II, and incubated with a dilution series ofthe Y•DTPA-DIG (small molecule) conjugate, followed by detection withanti-DIG Fab/AP and pNPP substrate (signal intensity is given inmOD/min). Recombinant wild type hNGAL revealed negligible signals inthis assay (not shown). Note that the hNGAL variant from the lastmaturation step, C26 (see inset), was immobilized at a significantlylower density (100 vs. 250 nM with a capture antibody concentration of2.5 vs. 10 μg/ml).

FIG. 2C shows the metal chelate binding activity of hNGAL variant C26 ina competitive ELISA. The setup of this ELISA was similar to the oneshown in panel (B), yet using a fixed concentration of theY•DTPA-RNase-DIG (protein) conjugate as tracer in the presence of avariable concentration of the free Me•Bn-CHX-A″-DTPA-Tris chelatecomplex or—for comparison—of Fc³⁺.enterobactin.

FIG. 2D depicts the kinetic real time analysis of hNGAL variant C26measured on a Biacore instrument. The Y•DTPA-RNase conjugate was coupledvia amine chemistry to a CM5 sensor chip (ΔRU=240) and the purifiedhNGAL variant C26 was applied at varying concentrations. The measuredsignal is shown as a grey line whereas the curve fit is depicted as ablack line in each case. The kinetic constants determined from this setof curves are listed in Table 3 (Example 13).

FIG. 2E depicts the kinetic real time analysis of hNGAL variant CL31measured on a Biacore instrument at a flow rate of 25 μl/min. TheY•DTPA-RNase conjugate was coupled via amine chemistry to a CM5 sensorchip (ΔRU=300), and the purified hNGAL variant CL31 was applied atvarying concentrations as indicated. The measured signal is shown as agray line and the fitted curve as a black line in each case.

FIG. 3 shows the crystal structure of the hNGAL variant Tb7.N9 incomplex with the Y•DTPA-Tris chelate with the polypeptide backbone isshown as ribbon in light grey whereas the bound Y³⁺•DTPA ligand is shownas a black stick model, including its 2F_(o)-F_(c) electrondensity—contoured at 1σ around the ligand DTPA and one Y³⁺-coordinatingwater molecule and at 4σ around the Y³⁺ ion. Within a 4 Å radiusaltogether 15 residues are found in contact distance with the boundmetal chelate complex, at least one in each of the eight β-strands:Gln33, Arg36, Thr52, Gln54, Val66, Ala68, Arg70, Asp77, Tyr78 (only viabackbone), Leu79, Met81, Phe83, Tyr106, Phe123, and Thr136. The sidechains of these residues are depicted as grey sticks, together withresidue Ser134 and a hydrogen-bonded water molecule that forms a bridgeto the metal-bound water.

FIG. 4 shows a potential application of hNGAL variants with Me•DTPAbinding activity for pretargeting radioimmunotherapy or in vivo imaging.FIG. 4 (A) shows a bispecific fusion protein or conjugate comprising (i)an NGAL mutein with Me•DPTA binding activity according to this invention(black) and (ii) an antibody/fragment or an alternative binding protein(e.g. another lipocalin mutein) with specificity for a tumor target isapplied to the blood stream. FIG. 4(B) shows the fusion proteinaccumulating at the tumor while unbound fusion protein is eliminated viathe kidney. FIG. 4(C): A radionuclide-DPTA complex is applied to thebloodstream. FIG. 4(D): The radionuclide-DTPA complex is bound by thetumor-associated fusion protein while excess complex is rapidly excretedvia the kidney. FIG. 4(E): Local decay of the bound radionuclide leadsto efficient cell death in the tumor, also taking advantage of abystander effect. FIG. 4(D*): Application of a bivalent version of theMe•DPTA complex leads to tighter binding at the tumor site via anavidity effect, thus allowing a prolonged retardation of theradionuclide.

EXAMPLES Example 1 Preparation of Me•DTPA Complex Conjugates

365 nmol (5 mg) bovine pancreatic ribonuclease A (RNase A; Fluka Chemie,Buchs, Switzerland), which exhibits up to ten Lys side chains as well asits free amino-terminus for covalent coupling, dissolved in 1 ml 100 mMNaHCO₃ (>99.5%; Carl Roth GmbH & Co, Karlsruhe, Germany), pH 8.3, wasreacted with a solution of 1.8 μmol (1.28 mg) p-SCN-Bn-CHX-A″-DTPA([(R)-2-amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaaceticacid.3HCl; Macrocyclics, Dallas, Tex.) in 10 μl DMSO over night at 4° C.under agitation. Typically, under these conditions one activated DTPAgroup reacted per protein molecule as quantified either by ESI-MS (QtoFUltima Global; Waters GmbH, Eschborn, Germany). Similarly, 150 nmol (10mg) bovine serum albumin (BSA; Sigma-Aldrich, Munich, Germany) wascoupled with 750 nmol (528 μg) p-SCN-Bn-CHX-A″-DTPA. For removal ofexcess reagent and buffer exchange a gel filtration on a PD-10 column(Amersham Pharmacia Biotech, Freiburg, Germany) was performed with 0.1 Mammonium acetate (>99.9%, Sigma-Aldrich)/acetic acid, pH 5 (Wu et al.,Bioorg Med Chem 5, 1925-1934 (1997). Then, an equimolar solution (withrespect to the carrier protein) of TbCl₃—or YCl₃, LuCl₃, GdCl₃, InCl₃(all from Sigma-Aldrich)—in the same ammonium acetate buffer was addedand, after incubation for 10 min at room temperature, the resultingconjugate was stored at −80° C. Using this procedure a Me•DTPA-RNaseconjugate with average 1:1:1 stoichiometry was obtained, as wasconfirmed by fluorescence titration of a sample of the gel-filtratedDTPA-RNase with the gravimetrically prepared TbCl₃ solution (λ_(Ex)=295nm, λ_(Em)=545 nm; FluoroMax-3; Jovin Yvon, Longjumeau, France),revealing a well detectable increase of Tb luminescence until saturationwas achieved.

A double conjugate of RNase (or BSA) with DTPA and digoxigenin (DIG) wasprepared by first reacting 915 nmol p-SCN-Bn-CHX-A″-DTPA in 10 μl DMSOwith 183 nmol of the carrier protein dissolved in 970 μl 100 mM NaHCO₃,pH 8.3, over night at 4° C. and then adding 366 nmoldigoxigenin-3-O-methylcarbonyl-ε-aminocaproic acid-N-hydroxy-succinimideester (DIG-NHS; Roche Diagnostics, Mannheim, Germany) in 20 μl DMSO forone hour at room temperature, followed by gel filtration and complexformation with the metal ion as above.

An Y-DTPA-Tris conjugate was prepared for co-crystallization with hNGALvariants by incubating 528 μg (750 nmol) p-SCN-Bn-CHX-A″-DTPA in 100 μL100 mM tris(hydroxymethyl)aminomethane (Tris; >99.9%, AppliChem,Darmstadt, Germany)/HCl, pH 8.0, over night at room temperature toachieve thiourea formation and adding 227 μg (750 nmol) Y³⁺, resultingin a final ligand concentration of 7.5 mM.

A direct conjugate of Me•DTPA with DIG-NHS was prepared by dissolving 2μmol p-NH₂-Bn-CHX-A″-DTPA([(R)-2-amino-3-(4-aminophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaaceticacid.4HCl; Macrocyclics) in 100 μl DMF with the addition of 1.7 μL (12μmol) diisopropylethylamine (Fluka) and reacting with 2 μmol DIG-NHSover night at room temperature. 10 μl of this solution was diluted with980 μl of the ammonium acetate buffer and 10 μl of 200 nmol YCl₃ orTbCl₃ in the same buffer was added.

Example 2 Construction of a Mutant hNGAL Phage Display Library

A combinatorial library of hNGAL variants was generated on the basis ofthe cloned cDNA (Breustedt et al. (2006) Biochim. Biophys. Acta 1764,161-173), which carried the amino acid substitutions Cys87Ser, to removethe single unpaired thiol side chain (Goetz et al. (2000) Biochemistry39, 1935-1941), as well as Gln28His and Thr145Ala to introduce twounique BstXI restriction sites with noncompatible overhangs, thuspermitting unidirectional cloning of the mutagenized central genecassette. Mutagenesis and polymerase chain reaction (PCR) assembly ofthis region was performed according to published strategy (Beste et al.(1999) Proc. Natl. Acad. Sci. USA 96, 1898-1903; Skerra (2001) J.Biotechnol. 74, 257-275) in two steps: First, two DNA fragments wereseparately amplified using pairs of degenerate oligodeoxynucleotides P1,5′-CAA TTC CAT GGG AAG TGG TAT YNS GTA GGT YNS GCA GGG AAT GCA NNS CTCAGA GAA GAC AAA GAC CCG CA-3′ (SEQ ID NO:11); and P2, 5′-GTG ACA TTG TAGCTC TTA TCT TCT TTC AGC TCA TAG ATS NRG GCS NNC ATC TTT TGC GGG TCT TTGTCT TC-3′ (SEQ ID NO:12); as well as P3, 5′-AAG AGC TAC AAT GTC ACA NNSGTC NNS TTT AGG AAA AAG AAG TGT GAC TAC NNS ATC NNS ACT TTT GTT CCA GGTTCC C-3′ (SEQ ID NO:13); and P4, 5′-GCC AGC TCC TTG GTT CTC CCS NRG AGSNRG ATS NNG AAG TAC TCC CTG TTT TGA G-3′ (SEQ ID NO:14), covering theamino acid positions 33/36/41, 52/54, 68/70/79/81, and 134/136/138,respectively. Second, both resulting PCR products were mixed in thepresence of the two flanking primers P5, 5′-CCA GGA CAA CCA ATT CCA TGGGAA GTG G-3′ (SEQ ID NO:15) and P6, 5′-GTT CCG AAG CCA GCT CCT TGG TTCTC-3′ (SEQ ID NO:16), followed by a few cycles of PCR for assembly andamplification of the full length central gene cassette. All PCR stepswere performed using Taq DNA polymerase (Fermentas MBI, St. Leon-Roth,Germany) as described (Schlehuber et al. (2000) J. Mol. Biol. 297,1105-1120). Oligodeoxynucleotides were purchased in HPLC grade fromThermo Fisher Scientific (Ulm, Germany) and further purified by ureaPAGE as necessary. The resulting DNA library was cut with BstXI(Promega, Mannheim, Germany) and cloned on the phagemid vector pNGAL35,which is based on the generic expression vector pASK75 (Skerra (1994)Gene 151, 131-135) and codes for a fusion protein composed of the OmpAsignal peptide, T7-tag, the modified mature hNGAL, followed by an ambercodon, and the C-terminal fragment of the gene III coat protein of thefilamentous bacteriophage M13, i.e. similar as previously described forthe bilin-binding protein (Beste et al., supra; Skerra, supra). Afterelectroporation of E. coli XL1-Blue (Bullock et al. (1987) Biotechniques5, 376-378) with the ligation mixture of 6 μg PCR product and 56 μgdigested plasmid DNA, ca. 6.5×10¹⁰ transformants were obtained.

Example 3 Selection of hNGAL Variants with Affinity to Metal ChelateComplex, Y•p-NH₂-Bn-CHX-A″-DTPA by Phage Display and Colony Screening

For production of recombinant phagemids, a culture of E. coli XL1-Bluetransformed with the pNGAL35 library was infected with VCS-M13 helperphages (Stratagene, Amsterdam Zuidoost, The Netherlands), wherebybiosynthesis of the hNGAL-pIII fusion protein was induced with 25 μg/Lanhydrotetracycline (Acros, Geel, Belgium) following published protocols(Beste et al., supra; Schlehuber et al., supra).

For each panning cycle about 10¹² recombinant phagemids in PBS (4 mMKH₂PO₄, 16 mM Na₂HPO₄, 115 mM NaCl, pH 7.4) were incubated for 1 h withImmunoSticks (Nunc, Wiesbaden, Germany) that had been coated with 100μg/ml of the TbDTPA-RNase conjugate and blocked for 2 h with 1.2 mlblocking buffer (PBS containing 0.1% (v/v) Tween 20 [polyoxyethylenesorbitan monolaurate; AppliChem] and 2% (w/v) BSA). After 8 washingsteps with PBS/T (PBS containing 0.1% (v/v) Tween 20), bound phagemidswere eluted for 15 min with 0.1 M glycine/HCl, pH 2.2, followed byimmediate neutralization with 0.5 M Tris base. The phagemids weretitered and reamplified prior to the next panning. After 7 cycles, anenrichment of the acid-eluted phagemids by a factor 1000 compared withthe phagemid number after the first cycle was observed.

Using the pooled phasmid preparation from the last panning step, themutagenized gene cassette was subcloned via BstXI on the plasmidpNGAL38, which encodes a fusion of the OmpA signal peptide, the hNGALcoding region with the C-terminal Strep-tag II (Schmidt and Skerra(2007) Nat. Protoc. 2, 1528-1535) followed by an amber stop codon aswell as a gene for the albumin-binding domain (ABD) from Streptococcalprotein G (Schlehuber et al., supra). Then, a filter sandwich colonyscreening assay was performed, whereby the hNGAL-ABD fusion proteins arereleased from the live colonies plated on a hydrophilic filter membraneand functionally captured on an underlying second membrane coated withhuman serum albumin (HSA) (Schlehuber et al., supra). This membrane wasprobed with 150 nM TbDTPA-BSA-DIG—or the corresponding RNaseconjugate—in PBS/T for 1 h, followed by development with an anti-DIGFab/alkaline phosphatase (AP) conjugate (Roche Diagnostics) andchromogenic staining according to the published protocol. Havingidentified spots with intense colour signals on this membrane, thecorresponding colonies were picked from the first filter and propagatedfor plasmid isolation and/or side by side comparison in a secondarycolony screen. During this step DTPA conjugates not charged with a metalion were used as negative control and, to avoid erroneous signalsarising from trace metal ion contamination, the high purity 0.1 Mammonium acetate buffer (>99.9%), pH 7.1, was employed.

For the subsequent improvement of affinity of the selected hNGAL variantTb7 (SEQ ID NO:2), the corresponding BstXI cassette was subjected toerror-prone PCR as described further below, followed by phagemiddisplay. In this case, 10¹¹ recombinant phagemids were incubated for 1 hwith ImmunoSticks that had been coated with 25 μg/ml of Tb•DTPA-RNaseconjugate for the first cycle and with 10 μg/ml of Tb•DTPA-RNaseconjugate for the second to fourth cycles.

For affinity maturation of the hNGAL variant Yd5 (SEQ ID NO:9), thecorresponding BstXI cassette was subjected to error-prone PCR (seebelow), followed by phagemid display, however, under conditions oflimiting off-rate. To this end, 10¹² phagemids were incubated for 1 h atroom temperature with ImmunoSticks that had been coated with 10 μg/ml ofY•DTPA-RNase conjugate. After 8 washing steps, the sticks were incubatedwith 800 μL of a 500 μM solution of the free metal chelate complex,Y•p-NH₂-Bn-CHX-A″-DTPA, in PBS for 30 min at room temperature to achievecompetition. After another 3 washing steps with PBS, remaining boundphagemids were eluted under acid conditions as above. In this case,three selection cycles were carried out in total.

For some of the affinity maturation steps, the mutagenized hNGALlibraries were directly applied to the colony screen, yet underincreasingly stringent conditions, by lowering the concentration of theTb/Y•DTPA-RNase-DIG conjugate from 50 nM to 5 nM. To raise thestringency even further, the strictly monovalent Tb/Y•DTPA-DIG smallmolecule conjugate at concentrations of 20 nM to 1 nM was applied.Finally, a competitive colony screen was performed by incubating thesecond membrane first for 1 h with 10 nM Y•DTPA-DIG and then, afterwashing three times with PBS/T, with 10 μM of the free complexYp-NH₂-Bn-CHX-A″-DTPA, followed by washing, detection, and staining asabove.

Example 4 hNGAL Mutagenesis by Error-Prone PCR

The construction of a second generation mutant library was carried outby PCR of the gene encoding Tb7 cloned on pNGAL15 with dNTP analogues(Zaccolo et al. (1996) J. Mol. Biol. 255, 589-603). The 20 μl reactionmixture contained 10 ng template DNA, 25 μM of each the dNTP analogues8-oxo-dGTP(8-oxo-2′-deoxyguanosine-5′-triphosphate) anddPTP(6-(2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-c][1,2]-oxazine-7-one-5′-triphosphate)(both from TriLink, San Diego, Calif.), 500 μM conventional dNTPs, 0.5μM flanking primers P5 and P6, 2 mM MgCl₂, and 2.5 units of Taq DNApolymerase. 10 cycles were carried out with temperatures of 92° C. for 1min, 55° C. for 1.5 min, and 72° C. for 5 min. Then reamplification wasperformed with 5 μl sample from above in 100 μl volume under the sameconditions but without the analogues using 20 cycles.

Randomization of the variant Yd5 was similarly performed by error-pronePCR using primers P5 and P6 in the presence of 50 μM dPTP, 50 μM8-oxo-dGTP, one unit of 9° N_(m) DNA polymerase (New England Biolabs,Frankfurt am Main, Germany) and followed by reamplification as above.The mutant 9° N_(m) DNA polymerase has 1-5% proofreading exonucleaseactivity in comparison with the wild type enzyme (Southworth et al.(1996) Proc. Natl. Acad. Sci. USA 93, 5281-5285) and was applied toenhance the transversion frequency.

The PCR products were purified by agarose gel electrophoresis (Sambrookand Russel, supra), cut with BstXI, and subcloned on pNGAL35 for phagedisplay selection.

Example 5 Targeted Random Mutagenesis of Amino Acid Subsets in the FirsthNGAL Variant

For randomization of positions 79 and 80, primers P5 (SEQ ID NO:15) andmut79back, 5′-GGA ACC TGG AAC AAA AGT CAT SNN SNN GTA GTC ACA CTT CTT-3′(SEQ ID NO:17), were applied in a PCR with Taq DNA polymerase as aboveusing pNGAL15-Tb7 as template. A second PCR fragment was generated usingprimers mut79for, 5′-GAC TTT TGT TCC AGG TTC C-3′ (SEQ ID NO:18), and P6(SEQ ID NO:16). Both fragments were assembled using the flanking primersP5 (SEQ ID NO:15) and P6 (SEQ ID NO:16) as described further above. Torandomize positions 125 and 127, primers P5 (SEQ ID NO:15) andmut127back, 5′-GCC AGC TCC TTG GTT CTC CCG AGG AGG GTG ATG GAG AAG TACTCC CTG TTT TGS NNA ACS NNC TTG AAG AAC ACC-3′ (SEQ ID NO:19), wereapplied in a PCR using pNGAL15-Tb7.N9 as template. The PCR product wasextended to full length via reamplification with primers P5 (SEQ IDNO:15) and P6 (SEQ ID NO:16). To randomize positions 77 and 136, primersP5 (SEQ ID NO:15) and mut77back, 5′-GGA ACC TGG AAC AAA AGT CAT GGT CAGGTA SNN ACA CTT CTT TTT CCT AAA CCT G-3′ (SEQ ID NO:20), were applied ina PCR using pNGAL15-Tb7.N9.N34 as template. A second PCR fragment wasgenerated using primers mut79for (SEQ ID NO:18) and mut136back, 5′-GCCAGC TCC TTG GTT CTC CCG AGG AGS NNG ATG GAG AAG TAC TCC CT-3′ (SEQ IDNO:21). Again, the two fragments were assembled using the primers P5 andP6 (SEQ ID NO:15 and 16).

To simultaneously randomize positions 33, 54, and 136, primers mut33for,5′-CAA TTC CAT GGG AAG TGG TAT NNS GTA GGT CGG GCA GGG-3′ (SEQ IDNO:22), and mut54back, 5′-CTT CTT TCA GCT CAT AGA TSN NGG CGG TCA TCTTTT GCG G-3′ (SEQ ID NO:23), were applied in a PCR usingpNGAL15-Tb7.N9.N34 as template. A second PCR fragment was amplifiedusing primers mutl36for, 5′-ATC TAT GAG CTG AAA GAA G-3′ (SEQ ID NO:24),and mut136back (SEQ ID NO:21). Again, both PCR fragments were assembledwith the flanking primers P5 (SEQ ID NO:15) and P6 (SEQ ID NO:16). Ineach case, the mutagenized DNA fragment was subcloned on pNGAL38 forsubsequent colony screen.

Example 6 Soluble Protein Production and Purification

The recombinant hNGAL and its variants were produced by periplasmicsecretion in E. coli BL21 (Studier and Moffat (1986) J. Mol. Biol. 189,113-130) or the supE strain TG1-F⁻ (a derivative of E. coli K12 TG1(Gibson (1984) Studies on the Epstein-Barr virus genome, CambridgeUniversity, England) that was cured from its episome using acridiniumorange with the plasmids pNGAL14 (Breustedt et al., supra) and pNGAL15for the wild type hNGAL and its variants, respectively, both encoding afusion of the OmpA signal peptide with the mature hNGAL protein and theC-terminal Strep-tag II, whereby the latter carries the twonon-compatible BstXI restriction sites for unidirectional subcloning ofthe mutated gene cassette. The soluble protein was affinity-purified bymeans of the Strep-tag II (Schmidt and Skerra, supra), followed by sizeexclusion chromatograpy (SEC) on a Superdex 75 HR 10/30 column(Amersham) using PBS buffer. Protein purity was checked by SDS-PAGE(Fling and Gregerson (1986) Anal. Biochem. 155 83-88) and proteinconcentrations were determined by absorption measurement at 280 nm usingcalculatory extinction coefficients of 29,930 M⁻¹cm⁻¹ for wtNGAL (SEQ IDNO:1) and of 21,680 M⁻¹cm⁻¹ for its variants Tb7 (SEQ ID NO:2), Tb7.14(SEQ ID NO:4), Tb7.N9 (SEQ ID NO:6), Yd5 (SEQ ID NO:9) and C26 (SEQ IDNO:10) (Gill and von Hippel (1989) Anal Biochem. 182, 319-326).

Example 7 Measurement of Binding Activity for the Me•DTPA Group in anELISA

For selective capturing of the hNGAL variants carrying the C-terminalStrep-tag II (Schmidt and Skerra, supra), a 96-well MaxiSorp polystyrenemicrotiter plate (Nunc) was coated with 50 μl of 5 to 10 μg/mLStrepMAB-Immo (IBA, Göttingen, Germany) in PBS over night at 4° C. andblocked with 1% (w/v) BSA in PBS/T at room temperature for 1 h. After 3washing steps with PBS/T, 50 μL of a 250 nM solution of the purifiedhNGAL variant was applied for 1 h to all wells. After washing, 50 μL ofa dilution series of the Me•DTPA-RNase-DIG conjugate was added andincubated for 1 h. The wells were washed again and bound conjugate wasdetected with 50 μL of anti-DIG Fab/AP conjugate diluted 1:1000 in PBSTfor 1 h, followed by signal development in the presence of 100 μl 0.5mg/ml p-nitrophenyl phosphate in 100 mM Tris/HCl, pH 8.8, 100 mM NaCl, 5mM MgCl₂. The time course of absorption ΔA/Δt at 405 nm was measured ina SpectraMax 250 reader (Molecular Devices, Sunnyvale, Calif.) and thedata were fitted with KaleidaGraph software (Synergy software, Reading,Pa.) to the equation

ΔA=ΔA _(max) ×[L] _(tot)/(K _(D) +[L] _(tot))

whereby [L]_(tot) represents the concentration of the applied ligandconjugate and K_(D) is the dissociation constant (Voss and Skerra (1997)Protein Eng. 10, 975-982). Alternatively, a competitive ELISA wasperformed in a similar manner, whereby the Me•DTPA-RNase-DIG conjugatewas applied at a fixed concentration of 2.5 to 5 nM in the presence ofvarying concentrations of the free Me•p-NH₂-Bn-CHX-A″-DTPA chelatecomplex in a range between 0.016 and 100 nM. In this case the data werefitted to the sigmoidal equation

ΔA=(ΔA _(max) −ΔA _(min))/(1+([L] _(tot) ^(free) /K _(D))^(p))+ΔAmin

with curve slope p (Hill coefficient) as a further parameter.

Alternatively, to further lower the concentrations of the stationaryassay components, a fluorimetric AP substrate was used. In this case, ablack Maxisorp 96-well microplate (Nunc) was coated with 50 μl of 5μg/ml StrepMAB-Immo, followed by a 100 nM solution of the purified hNGALvariant and a fixed concentration of 2.5 nM Y•DTPA-RNase-DIG wasapplied. Signals were developed by adding 50 μl of 1 mM AttoPhos (Roche)in a buffer supplied by the manufacturer. Kinetic fluorescencemeasurements were made on a FluoroMax-3 microplate reader (λ_(Ex)=430nm, λ_(Em)=535 nm) and evaluated as above.

Example 8 Measurement of Binding Activity for the Me•DTPA Group ViaSurface Plasmon Resonance (SPR)

Real time analysis of hNGAL variants was performed on a BIAcore X system(BIAcore, Uppsala, Sweden) using PBS/t (PBS containing 0.005% (v/v)Tween 20) as running buffer. 5 to 27 μg/ml solutions of theMe•DTPA-RNase conjugate in 10 mM Na-acetate, pH 5.0 were immobilized ona CM5 chip using standard amine coupling chemistry, resulting in aligand density of 240 to 1800 resonance units (RU). The purified hNGALvariant was applied at a flow rate of 5 or 25 μl/min at concentrationsof 0.5 up to 500 nM. The sensorgrams were corrected by subtraction ofthe corresponding signals measured for the control channel, which hadbeen activated and blocked with ethanolamine. Kinetic data evaluationwas performed by global fitting with BIAevaluation software V 3.0(Karlsson et al. (1991) J. Immunol. Methods 145, 229-240).Alternatively, the plateau values at the end of the association phase(after 200 s) were plotted against the applied protein concentration andfitted as in the ELISA (see above) to determine the equilibriumdissociation constants (K_(D)).

Example 9 Crystallization of hNGAL Variants

After dialysis against 100 mM NaCl, 10 mM Tris/HCl, pH 8.0 the hNGALvariants Tb7.14 (SEQ ID NO:4) and Tb7.N9 (SEQ ID NO:6) were concentratedto 18 and 25 mg/ml, respectively, using 10 kDa cut-off Ultrafreeconcentrators (Millipore, Billerica, Mass.) and sterile filtered with a0.45 mm Costar Spin-X centrifuge unit (Corning, Corning, N.Y.). Bothproteins were crystallized using the hanging drop vapour-diffusiontechnique (Mc Pherson, Crystallization of biological macromolecules,Cold Spring Harbor, N.Y. Cold Spring Harbor Larboratory Press). Forcrystallization of Tb7.14, 1 μl solution of the apo-protein was mixedwith 1 μl reservoir solution, comprising 2 M (NH₄)₂SO₄, 200 mM Li₂SO₄,100 mM Tris/HCl, pH 7.0. Crystals of space group P4₁2₁2 with threemolecules per asymmetric unit were obtained after 6 weeks at 20° C.Tb7.N9 (SEQ ID NO:6) was crystallized at a final protein concentrationof 22 mg/mL (1.1 mM) after adding a slight excess of Y•DTPA-Tris (1.6mM). In this case 1 μl protein/ligand solution was diluted with 1 μlwater and mixed with 1 μl reservoir solution, comprising 22% (w/v) PEG3350, 100 mM Bis-tris/HCl, pH 5.5. Crystals of space group P4₁2₁2 withtwo protein chains per asymmetric unit were obtained after one week at20° C. Crystals of the two hNGAL variants were soaked in thecorresponding precipitant solution supplemented with 30 and 20% (v/v)glycerol, respectively, prior to freezing in liquid nitrogen.

Example 10 Data Collection and Model Building

Crystal diffraction data for the Tb7.14 (SEQ ID NO:4) apo-protein andthe Tb7.N9 (SEQ ID NO:6)-ligand complex were collected at BESSY (Berlin,Germany) beamlines 14.1 and 14.2, respectively (Table 1).

TABLE 1 Data collection and refinement statistics Dataset Tb7.14Tb7.N9/Y · DTPA- Tris Space group P4₁2₁2 P4₁2₁2 Unit cell dimensions113.59, 113.59, 119.79 82.35, 82.35, 115.13 a, b, c [Å], α = β = γ = 90°molecules/asymmetric unit 3 2 Wavelength [Å] 0.95373 0.95373 Resolutionrange [Å]^(a) 40.00-2.50  40.00-2.00  (2.64-2.50) (2.11-2.00) I/σI 2.8(2.0) 4.1 (2.0) R_(merge) [%]^(a) 15.4 (33.1) 10.9 (37.2) Uniquereflections 27785 26827 Multiplicity 8.7 (8.9) 9.7 (9.8)Completeness^(a) 100.0 (100.0) 98.2 (97.4) Refinement:R_(cryst)/R_(free) 25.56/30.46 21.31/23.38 Protein atoms 4081 2772Ligand atoms — 98 Ion atoms — 2 Solvent atoms 480 327 Average B-factor[Å²] 36.50 27.62 Geometry: R.m.s.d. bond lengths/ 0.0084/1.44 0.0094/1.83  angles [Å/deg] Ramachandran analysis: Core, allowed,generously 85.6, 10.9, 1.6, 1.8 90.5, 7.8, 0.3, 1.4 allowed, disallowed[%]

The data were processed with MOSFLM, scaled with SCALA, and reduced withTRUNCATE (CCP4 (1994) Acta Crystallogr. D 50, 760-763). Molecularreplacement of the Tb7.14 apo-protein was carried out with the programEPMR (Kissinger et al. (2001) Acta Crystallogr. D Biol. Crystallogr. 57,1474-1479) using the structure of the wild type siderocalin hNGAL withall three molecules of the asymmetric unit (Holmes et al. (2005)Structure 13, 29-41) (PDB entry 1X71). Molecular replacement of theliganded Tb7.N9 (SEQ ID NO:6) was subsequently carried out with theprogram MOLREP (CCP4, supra) using the refined model of Tb7.14 (SEQ IDNO:4).

The atomic models were built with the program 0 (Jones et al. (1991)Acta Crystallogr. A 47, 110-119). For Tb7.14 (SEQ ID NO:4), there waselectron density—at a resolution of 2.5 Å—for residues Ser3-Leu42,Pro48-Gly178 of molecule 1, Ser3-Ala41, Gln49-Asp177 of molecule 2, andThr4-Leu42, Pro48-Asp177 of molecule 3. For Tb7.N9 (SEQ ID NO:6), therewas electron density at a resolution of 2.0 Å—for residues Asp6-Ala40,Arg43-Asp177 of molecule 1 and residues Leu7-Ala40, Leu42-Gly178 ofmolecule 2. The ligand Y•DTPA-Tris was modelled using Insight II(Accelrys, San Diego, Calif.) on the basis of the crystal structure ofan In•DTPA complex (CSD entry MOQVOD), and corresponding topology andrefinement parameters were generated using PRODRG (Schuttelkopf and vanAalten (2004) Acta Crystallogr. D Biol. Crystallogr. 60, 1355-1363).Both crystal structures were refined with CNS (Brünger et al. (1998)Acta Crystallogr. D Biol. Crystallogr. 54, 905-921) and water moleculeswere added. Rotamers of asparagine and glutamine residues were checkedwith NQ-Flipper (Weichenberger and Sippl (2006) Bioinformatics 22,1397-1398). The refined structural models were validated with PROCKECK(Laskowski et al. (1993) J. Appl. Cryst. 26, 283-291) and WHAT_CHECK(Hooft et al. (1996) Nature 381, 272). Secondary structure elements wereassigned using DSSP (Kabsch and Sander (1983) Biopolymers 22, 2577-2637)and protein-ligand contact surfaces were calculated with PISA (Krissineland Henrick (2007) J. Mol. Biol. 372, 774-797). Molecular graphics andstructural superpositions were made with PyMOL (DeLano (2002) The PyMOLMolecular Graphics System, DeLano Scientific, San Carlos, Calif., USA)while the protein-ligand interactions diagram was prepared with LIGPLOT(Wallace et al. (1995) Protein Eng. 8, 127-134).

Example 11 Selection of Lipocalin Variants with Me•DTPA Specificity froma Lipocalin Random Library

Based on the known crystal structure of hNGAL in complex with itsnatural ligand enterobactin (Goetz et al., supra) and its cloned cDNA(Breustedt et al., supra), we constructed a combinatorial library byspecifically randomizing the codons for 12 amino acid positions in thebinding pocket (FIG. 1, Table 2).

Residues at the bottom of the ligand pocket and in close proximity tothe natural iron siderophore complex, including two of the threepositively charged side chains R81, K125, and K134 (Goetz et al.,supra), were preferentially chosen for the targeted mutagenesis. Thesepositions were expected to tolerate both small and large side chainsubstitutions, reaching as deeply as possible into the cavity withoutaffecting the hydrophobic residue packing in the lower part of theβ-barrel.

TABLE 2  Amino acid sequences of selected hNGAL variants. hNGAL Tb7Tb7.14 Tb7.N9 Tb7.N9.N34 Ya6 Yd5 C26 Residue (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID No.^(a) NO: 1) NO: 2) NO: 4)NO: 6) NO: 7) NO: 8) NO: 9) NO: 10)  ^(a)28 Gln His His His His His HisHis  ^(a)87 Cys Ser Ser Ser Ser Ser Ser Ser ^(a)145 Thr Ala Ala Ala AlaAla Ala Ala   33 Val Gln Gln Gln Gln Gln Gln Gln   36 Leu Arg Arg ArgArg Arg Arg Arg   41 Ile Ala Ala Ala Ala Ala Ala Ala   52 Tyr Thr ThrThr Thr Thr Thr Thr   54 Thr ^(b)Gln Gln Gln Gln Gln Gln Gln   68 SerAla Ala Ala Ala Ala Ala Ala   70 Leu Arg Arg Arg Arg Arg Arg Arg   79Trp Ala Ala Leu Leu Leu Leu Leu   81 Arg Met Met Met Met Met Met Met 134 Lys Ser Ser Ser Ser Ser Ser Ser  136 Thr Thr Thr Thr Thr Ser SerSer  138 Tyr Leu Leu Leu Leu Leu Leu Leu  ^(c)77 Asp Asp Asp Asp Asp AspGlu Glu  ^(c)80 Ile Ile Thr Thr Thr Thr Thr Thr ^(c)127 Ser Ser Ser SerGln Gln Gln Gln  ^(d)42 Leu Leu Leu Leu Leu Leu Leu Pro  ^(d)48 Pro ProPro Pro Pro Pro Pro Leu  ^(d)49 Gln Gln Gln Gln Gln Gln Gln Leu  ^(d)55Ile Ile Ile Ile Ile Ile Ile Thr ^(a)Sequential numbering of the matureprotein sequence (cf. SwissProt entry P80188). Positions 28, 87, and 145were specifically mutated for reasons of genetic manipulation. ^(b)Theamber stop codon is translated as Gln in the supE background of thebacterial strains that were used for the selection experiments and waslater replaced by the codon CAG. ^(c)Accidental mutations at positionsthat were not part of the initial random mutagenesis. ^(d)Mutationsarising from error-prone PCR with the nucleotide analogues and 9° N_(m)DNA polymerase.

This strategy could be expected to allow efficient reshaping of theligand pocket to achieve a novel specificity for the smaller DTPA metalchelate complex, similarly as it was previously demonstrated with aninsect lipocalin and organic molecules as ligands (Beste et al., supra;Schlehuber et al., supra).

Concerted random mutagenesis of these positions, which were spreadacross large part of the hNGAL primary sequence, was realized accordingto a previously developed PCR assembly strategy (Beste et al., supra;Skerra (2001), supra) with appropriate modifications. To this end, twogene segments, each comprising one pair of the altogether four clustersof randomized residues (#1: 33, 36, 41; #2: 52, 54; #3: 68, 70, 79, 81;#4: 134, 136, 138), were first separately amplified, usingoligodeoxynucleotides with degenerate NNS codons at the desiredpositions. The two resulting PCR products which included a short overlapin the middle of the NGAL gene were isolated, mixed, and then assembledin another amplification just with few cycles, using flanking primersthat contributed two unique BstXI restriction sites (see above). Afterunidirectional cloning on a suitable phasmid vector for filamentousphage display (Skerra (2001), supra), a molecular library with adiversity of approximately 6.5·10¹⁰ independent transformants wasobtained.

This library was employed for the enrichment of Me•DTPA specific hNGALvariants via panning on ImmunoSticks coated with the immobilized ligand.For this purpose, a ligand derivative with a chemically reactiveisothiocyanate group, p-SCN-Bn-CHX-A″-DTPA, was covalently coupled toRibonuclease A, which served as a robust carrier protein devoid ofnon-specific binding activities (Schlehuber et al., supra), and chargedwith the transition metal ion Tb³⁺. This lanthanide was chosen as amodel ion for the initial selection experiments as it (i) shows aluminescent behaviour strongly dependent on its molecular environment(Martini et al. (1993) Eur. J. Biochem. 211, 467-473; Corneillie et al.,supra; Handl and Gillies (2005) Life Sci. 77, 361-371), which washelpful to analyze the proper charging of the protein-DTPA conjugate,and (ii) its radius is not too much different from therapeutically ordiagnostically relevant radioactive isotopes such as ⁹⁰Y³⁺, ¹¹¹In³⁺ and¹⁷⁷ _(Lu) ³⁺ (Goldenberg (2002) J. Nucl. Med. 43, 693-713; Boerman etal. (2002) J. Nucl. Med. 44, 400-411; Corneillie et al., supra; Kenanovaand Wu, supra).

After seven cycles of phagemid display panning, the enriched pool ofhNGAL variant genes was subcloned on another plasmid and subjected to afilter sandwich colony screen (Schlehuber et al., supra). In thisexperiment the hNGAL variants became secreted from live E. coli coloniesas a fusion with a bacterial albumin-binding domain (ABD) and wereimmediately bound to an underlying filter membrane coated with humanserum albumin (HSA). The functionally immobilized hNGAL variants wereprobed for binding of an RNase double conjugate with Tb•DTPA anddigoxigenin groups. After signal development with an anti-DIG Fab/APconjugate several clones with specific ligand-binding activity wereidentified. Sequence analysis of 16 selected clones revealed that 7 ofthese had an identical sequence, which was named Tb7 (SEQ ID NO:2)(Table 2), whereas the remaining clones showed very similar sequences,with up to 4 amino acid exchanges compared with Tb7.

Example 12 Affinity and Specificity of Selected hNGAL Variants for theMe•DTPA Chelate Complex

The hNGAL variants were subcloned onto a suitable expression vector andproduced as soluble proteins in the periplasm of the E. coli strainBL21, which lacks endogenous enterobactin (Goetz et al., supra).Purification from the periplasmic protein extract by means of theC-terminal Strep-tag II (Schmidt and Skerra, supra) and size exclusionchromatography yielded 0.5-3 mg protein per 1 L shake flask culture,similarly as for the recombinant wild type protein (Breustedt et al.,supra). The purity was greater than 95% as determined by Coomassiestained SDS-PAGE (FIG. 2).

Binding activity was first investigated in an EISA for the Tb•DTPA-RNaseA conjugate that had also served during the selection procedure (seeMaterials & Methods). The variant Tb7 showed a strong andmetal-dependent signal (FIG. 2B) with an apparent affinity in the lownanomolar range (74.5±7.8 nM) whereas wild-type hNGAL exhibited nomeasurable binding activity.

Example 13 Affinity Maturation of hNGAL Variant Tb7 for Improved Me•DTPABinding

Starting with the coding region for the hNGAL variant Tb7, a secondgeneration library was constructed by error-prone PCR of the centralgene cassette (flanked by the two BstXI restriction sites at amino acidpositions 25-29 and 141-145) in the presence of deoxynucleotideanalogues (Zaccolo et al, supra), followed by four cycles of phagemiddisplay and colony screening under more stringent conditions (cf.above). DNA sequencing of 10 clones from the colony screen giving riseto the most intense signals for Tb•DTPA binding showed that nine of themcarried the amino acid substitution Ile80Thr, together with one or twoadditional substitutions at positions 65, 71, 73, 74, 75, 116 or 135.The three variants Tb7.1 (Lys75Asp/Ile80Thr) (SEQ ID NO:3), Tb7.14(Ile80Thr) (SEQ ID NO:4), and Tb7.17 (Phe71Ser/Lys73Glu/Ile80Thr) (SEQID NO:5) were again expressed as soluble proteins in E. coli BL21. Afterpurification as above, their ligand-binding properties were investigatedin an ELISA (data not shown). These three variants exhibited somewhathigher signal intensities at saturation than Tb7 but their half-maximalconcentration values for binding of the Tb-DTPA complex were about 15 nMand, thus, not much improved.

In the second stage, saturation mutagenesis was performed in aconsecutive manner for the amino acid sets at positions 79/80, 125/127,and 77/136 as well as 33/54/136. Positions were chosen according to themutational patterns of promising variants identified so far and by usingmolecular model building on the basis of the wild type hNGAL structureas well as the crystal structure of its variant Tb7.14 (SEQ ID NO:4; seebelow). To this end, a PCR assembly strategy similar to the one for theconstruction of the original library was applied, employing pairs ofsynthetic oligodeoxynucleotides carrying fully degenerate codon (oranticodon) sequences at the desired positions (cf. above). As theresulting molecular libraries had low combinatorial complexities theywere directly subjected to the colony screening assay as before, howeverusing lower ligand concentration during the selection. For mutagenesisof positions 77/136 and 33/54/136, the Y•DTPA-DIG conjugate was appliedas target (i.e. switching from Tb³⁺ to the medically more relevant Y³⁺),whereas selection at positions 79/80 and 125/127 was performed withTb•DPTA-RNase-DIG conjugate.

After each random mutagenesis the best resulting clone was chosenaccording to its ligand affinity, yield of soluble periplasmicexpression, and stable monomer formation during gel filtration ascriteria. During this procedure three promising variants with improvedbinding activities (up to app. 30-fold) subsequently emerged: Tb7.N9(A79L/I80T) (SEQ ID NO:6), Tb7.N9.N34 (S127Q) (SEQ ID NO:7), and Yd5(D77E/T136S) (SEQ ID NO:9) (Table 2). The final variant Yd5 showed thebest binding activity, with a K_(D) value of 3.5 nM (see below and Table3).

TABLE 3 The dissociation constants and binding kinetics of selectedhNGAL variants for Y³⁺ · DTPA-RNase/Tb³⁺ · DTPA-RNase measured byreal-time SPR analysis at a flow rate of 5 μl/min. Y³⁺ · DTPA-RNaseLipocalin mutein k_(on) [M⁻¹ s⁻¹] k_(off) [s⁻¹] K_(D) [nM]^(a) τ_(1/2)[s] Tb7 9.98 × 10⁵ 0.1 100 6.9 Tb7.N9 1.61 × 10⁶  2.3 × 10⁻² 14.3 30Tb7.N9.N34 1.57 × 10⁶ 2.08 × 10⁻² 13.2 33 Ya6 1.72 × 10⁶ 5.53 × 10⁻³ 3.2125 Yd5 9.84 × 10⁵ 3.45 × 10⁻³ 3.5 201 C26  2.5 × 10⁶ 7.04 × 10⁻⁴ 0.282984 Tb³⁺ · DTPA-RNase Lipocalin K_(D) τ_(1/2) mutein k_(on) (M⁻¹ s⁻¹)k_(off) (s⁻¹) [nM]^(a) K_(d/eq) [nM]^(b) (s) Tb7 5.35 × 10⁵ 4.01 × 10⁻²74.9 50 ± 6.5 17 Tb7.N9 6.89 × 10⁵ 1.05 × 10⁻² 15.2 25 ± 2.7 66Tb7.N9.N34 7.46 × 10⁵ 1.14 × 10⁻² 15.3 21 ± 2.3 61 Ya6   1.24 × 10⁻⁶2.96 × 10⁻³ 2.39  5 ± 0.22 234 Yd5   1 × 10⁶ 2.46 × 10⁻³ 2.34  3.8 ±0.15  282 ^(a)determined from the kinetic analysis ^(b)determined fromthe concentration-dependent saturation values in an equilibrium analysisData analysis was performed by global fit according to the 1:1 bindingmodel

In the third stage, another error prone PCR mutagenesis of the wholecentral coding cassette was performed on the basis of Yd5 (SEQ ID NO:9)and followed by phage display selection towards slow dissociationkinetics by using competitive conditions. To this end, panning wasperformed with the Y•DTPA-RNase target adsorbed to ImmunoSticks and,after washing, bound phagemids were incubated in the presence of a 500μM solution of the free metal chelate complex, Y•p-NH₂-Bn-CHX-A″DTPA, ascompetitor for 30 min, followed by another three washing steps and,finally, acid elution. After three cycles of phagemid selection, theenriched pool of hNGAL variants was subcloned and subjected to thecolony screening assay, again applying competitive conditions. 16variants giving rise to intense staining signals, even in the presenceof an approximately thousand-fold molar concentration of the unlabelledligand, were sequenced. Thus, the variant C26 (SEQ ID NO:10) wasisolated, which exhibits five additional mutations(L42P/P48L/Q49L/I55T/K75M), most of them in loop #1.

Example 14 Biochemical Characterization of Selected hNGAL Variants byCompetition ELISA and Surface Plasmon Resonance

The hNGAL variants resulting from the affinity maturation of Tb7 wereproduced in E. coli as soluble proteins at the shake flask scale andpurified via the Strep-tag II and SEC as before. All selected variantsexhibited excellent expression characteristics and were purified asstable and fully monomeric proteins (FIG. 2A) from the periplasmic cellextract, showing final yields of 0.5 to 3 mg per liter shake-flaskculture.

Binding activities were first compared in an ELISA using the hNGALvariants captured to the microtiter plate and incubating them withvarying concentrations of the Y•DTPA-DIG conjugate, which was detectedwith an anti-DIG Fab/AP conjugate (FIG. 2B). All variants, starting fromTb7 to its most recently improved derivatives, showed hyperbolicsaturation curves whereas wild type hNGAL did not reveal any bindingactivity for the metal chelate complex (not shown). The amplitude of thesaturation curves increased while the half-maximal ligandconcentration—corresponding to apparent K_(D) values of 74±8 nM (Tb7),10.8±1.2 nM (Tb7.N9), 5.0±0.7 nM (Yd5), 0.43±0.06 nM (C26)—decreasedover the course of the in vitro affinity maturation.

The binding activity of the hNGAL variants for the small soluble chelateligand was further investigated in a competition ELISA using microtiterplates coated with Tb•DTPA-RNase and Me•p-NH₂-Bn-CHX-A″-DTPA as freeligand competitor, using Tb³⁺ and Y³⁺ as well as other trivalent metalions. These measurements showed nice inhibition curves for theTb³⁺/Y³⁺-charged chelate complex especially in case of the variantsTb7.N9, Yd5, and C26 resulting from the affinity maturation. Incontrast, the binding activity of Tb7 for the immobilized ligand wasprobably too weak to yield a proper competition effect. The finalvariant, C26, was more thoroughly analyzed using different metal ions(FIG. 2C). Its K_(D) values deduced from the half-maximal free metalchelate concentrations were: 2.7±0.03 nM (Y³⁺), 3.6±0.24 nM (Gd³⁺),2.9±0.17 nM (Tb³⁺), 9.4±0.33 nM (Lu³⁺), 44.7±2.5 nM (In³⁺), and 95±7 nM(Bi³⁺). Thus, the engineered hNGAL variants, especially C26, exhibitstrong binding activity towards the small metal chelate ligand whereasthe context of RNase, which was employed as carrier protein during theselection, does not play a significant role.

Finally, the hNGAL variants were analyzed by SPR using a Biacore CM-5chip with the covalently attached Y-DTPA-RNaseA conjugate and applyingthe purified recombinant proteins (Table 3). Again, wild-type hNGAL didnot exhibit any significant binding activity whereas moderate bindingsignals were obtained for Tb7 (not shown). However, its derivativesisolated at different stages of the in vitro affinity maturation showedincreasing affinity towards the immobilized target, with higher valuesaround 3 nM for Ya6 and Yd5. This represents an approximately 30-foldimprovement over the parental lipocalin mutein Tb7 (K_(D)=75 to 100 nM).The improved K_(D) values result primarily from slower liganddissociation. In this respect the two variants with higher affinitiesdiffer, whereby Yd5 shows a significantly longer half life ofdissociation (ca. 200 s). Notably, the variant, C26 resulting from thecompetitive selection experiment revealed even a 282 pM affinity to themetal chelate ligand (FIG. 2D).

Example 15 Crystallographic Analysis of hNGAL Variants with Me•DTPABinding Activity

The hNGAL variants Tb7.14 (SEQ ID NO:4) and Tb7.N9 (SEQ ID NO:6) weresubjected to X-ray crystallographic analysis, the latter in complex withthe ligand Y•DTPA-Tris. As expected, both proteins exhibit the typicallipocalin fold comprising an eight-stranded antiparallel β-barrel withan α-helix attached to its side (FIG. 3). Superposition of the 58mutually equivalent backbone positions of Tb7.14 and of Tb7.N9 (Cα atoms28-37, 52-58, 63-69, 77-84, 91-94, 106-113, 118-124, 133-139, each fromchain A), which are structurally conserved for the β-barrel of thelipocalins (Skerra (2000), supra), resulted in a root mean squaredeviation (r.m.s.d.) of 0.31 Å. The r.m.s.d. for the chains A of Tb7.14and of hNGAL in complex with trencam-3,2-hopo (PDB entry 1X71), whichhad been used for molecular replacement, was 0.24 Å (for Cα atoms 7-40and 49-177) while the mutual r.m.s.d. between chains A/B and A/C in theasymmetric unit of Tb7.14 was 0.43 Å and 0.35 Å, respectively. For chainA of Tb7.14 and chain A of hNGAL in complex with enterobactin (PDB entry1L6M) the r.m.s.d. was 0.26 Å while the corresponding r.m.s.d. forTb7.N9 was slightly higher with 0.31 Å. The mutual r.m.s.d. betweenchains A and B in the asymmetric unit of Tb7.N9 was 0.58 Å. A comparisonof the side chain conformations of Tb7.14 and Tb7.N9 showed majorchanges only for residues Gln33, Arg36, Gln49, Gln54, and Thr136. Theside chain of Arg36 forms a hydrogen bond between its guanidinium NH1atom and a carboxylate oxygen (O2) of the bound DTPA (3.3 Å).

In the complex with Tb7.N9 the bound Y•DTPA-Tris nestles at one side ofthe cleft at the open side of the β-barrel and fills about one third ofits volume. The remainder of the cavity is occupied with 9 watermolecules, which form a hydrogen bond network. There are no directcontacts between amino acids and the lanthanide ion. The DTPA derivativefills eight of nine coordination sites of Y³⁺ while the ninth site isoccupied by a water molecule (HOH9). This water molecule is in hydrogenbond distance with two of the DTPA carboxylate groups (HOH9-LIG O1, 2.9Å; HOH9-LIG O9, 3.0 Å) and with another crystallographically definedwater molecule (HOH143, 2.6 Å distance)—in the second shell around themetal ion which itself is hydrogen-bonded to Ser134 (HOH 143-Ser134 OG,2.9 Å).

The entire chelate complex is oriented with its hydrocarbon groups,including the cyclohexane ring and the benzyl side chain, towards acontiguous hydrophobic stretch on β-strands B, C, and D (FIG. 3). Thepolar carboxylate groups, including the Y³⁺-coordinating water molecule,point towards β-strands G, H, and A, where a gap filled with watermolecules is formed, which was previously occupied by the naturalsiderophore in the case of wild type hNGAL.

The DTPA part of the ligand, which is nicely defined in the electrondensity revealing the anticipated chirality (Brechbiel and Gansow (1992)J. Chem. Soc. Perkin Trans 1, 1173-1178), can be described as a baseballglove with the metal ion representing the grabbed baseball, similar asit was previously seen in the small molecule crystal structure of anIn³⁺•DTPA complex (Maecke et al. (1989) J. Nucl. Med. 30, 1235-1239).The Y³⁺ ion is coordinated by nine atoms. Eight of them stem from theoctadentate chelating ligand, five from its carboxylate oxygens(distances 2.3-2.5 Å) and three from its amine nitrogens (distances2.5-2.7 Å), whereas one is a bound water oxygen (distance app. 2.7 Å).Similarly as in the natural hNGAL enteroactin complex, there are nodirect liganding contacts between the metal ion and protein side chains.The thiourea group protruding from the benzyl side chain of the DTPAderivative and the conjugated Tris moiety are orientated outwards fromthe lipocalin cleft. There are two hydrogen bonds between atom N4 of thethiourea group and the two carboxylate oxygens of Asp77, (OD1: distance3.4 Å; OD2: distance 2.9 Å). The terminal tris-hydroxymethyl group isonly partially defined in the electron density (FIG. 3).

Structural superposition of the individually refined protein chains fromthe crystal structures of apo-Tb7.14 and of the Tb7.N9 Y•DTPA-Triscomplex with wild type hNGAL in complex with enterobactin shows thatdespite the large number of 16 amino acid exchanges the overall fold isextremely well conserved. In particular, the β-barrel itself, the shortloops at its closed end, the α-helix attached to its side, and even themore or less flexible N- and C-terminal extensions of the polypeptidechain are almost indistinguishable (FIG. 3). The set of 58 Cα positionsthat are structurally conserved among the lipocalin family (Skerra(2000), supra), show r.m.s.d. values of 0.256, 0.440, 0.290, 0.371, and0.386 Å, respectively, although the three proteins were crystallized innon-isomorphic space groups and, thus, with different crystal packingneighborhood.

Even more surprising, the set of four loops at the open end of theβ-barrel, which harbors most of the side chain substitutions that wereintroduced to reshape the ligand pocket for the binding of DTPA havelargely retained their geometry compared with the wild type protein.Especially loops #2, #3, and #4—connecting β-strands C/D, E/F, andG/H—exhibit an unchanged conformation; except for individual minorshifts of the loops as a whole. A maximum shift of ca. 1.2 Å is seen forthe Cα position 73 at the tip of loop #2 in chain C of the apo-Tb7.14structure, which is likely to reflect a crystal packing effect.

In contrast, the rather long Ω-type loop #1 shows considerable backboneflexibility among the three different crystal structures. Especially forchains A and C of the apo-Tb7.14 structure it is almost identical withwild type hNGAL in the region of residue 46, while significantdeviations occur around residue 41. In the case of chain B, however, theentire segment between residues 40 and 49 is shifted by almost 5 Å (forthe backbone). In case of the two chains of the Tb7.N9 DTPA complex thisshift is even more severe and individually different, whereby thereappears one turn of a 3₁₀ helix around residue 44 for chain B. Notably,there is no electron density for residues 43-47 (chain A), 42-48 (chainB), and 43-47 (chain C) of this loop in the apo-Tb7.14 X-ray structureand this loop does not form crystal contacts in this structure as wellas in the one of the DTPA complex. Hence, the conformation of loop #1seems not only to be influenced both by the molecular environment butalso by the presence of the metal chelate ligand. This behavior suggestthat future mutational studies should be focused at loop #1 in order toachieve altered backbone conformations that lead to closer interactionwith the bound DTPA ligand and, possibly, to even higher affinities.

Y•DTPA-Tris is bound more deeply than the natural ligand enterobactin atthe bottom of the hNGAL cavity and almost situated in the mid among the12 residues that were randomized in the original random library. Thediethylenetriamino moiety interacts mainly with residues on β-strands Cand D, whereby the cyclohexane ring packs against the hydrophobicresidues Val66, Leu79, and Met81 while the benzyl side chain issandwiched between Arg70 and Leu79 and protrudes with its Trissubstituent into the solvent. The entire cavity is positively charged,similarly to wild type hNGAL (Goetz et al., supra), with the exceptionof the stretch of the residues Val111, Val121, and Phe123 at the bottomof the cavity. Notably, Met81 has replaced Arg71 in hNGAL, which is oneof the three positively charged side chains that were described toparticipate in cation-π interactions with the bound enterobactin (Goetzet al., supra). Lys134 is replaced by Ser, thus providing space for themetal ion as well as two of the DTPA carboxylate groups and theliganding water molecule, and the more remote residue Lys125 maycontribute to a general electrostatic interaction with the overallnegatively charged metal chelate complex.

The larger side chain of residue Leu at position 79 compared with theone of Ala in the variant Tb7.14 leads to an improved van der Waalscontact to the phenyl thiourea group of DTPA. The additional hydrogenbond between OG1 of the new side chain Thr80 (cf. Table 2) and OG1 ofThr67 locally stabilizes the pairing of strands C/D on the outside ofthe β-barrel. The buried surface area of the Y•DTPA-Tris amounts to 586Å², which is about the same buried surface as for enterobactin bound tohNGAL.

Within a 4 Å radius altogether 15 residues are found in contact distancewith the bound metal chelate complex, at least one in each of the eightβ-strands: Gln33, Arg36, Thr52, Gln54, Val66, Ala68, Arg70, Asp77, Tyr78(only via backbone), Leu79, Met81, Phe83, Tyr106, Phe123, and Thr136.Despite the smaller size of the ligand compared with enterobactin thisis possible due to its deeper burial within the lipocalin cavity. On theother hand, there are rarely any contacts with the four loops. Amongthese residues, 9 positions were subject to mutagenesis in the initialhNGAL random library (cf. Table 2). The substitution Asp77Glu was foundat a later stage of affinity maturation, whereas only four of thecontacting side chains 66, 83, 106, and 123 correspond to originalresidues of hNGAL. Interestingly, they still exhibit the same rotamersin the Y•DTPA complex. Apart from these minor remnants, the mode ofbinding is totally different for the Y³⁺•DTPA complex in the case of theengineered lipocalin than for Fe³⁺.enterbactin by hNGAL.

Even though the two crystal structures were obtained for hNGAL variantsat intermediate stages of the affinity maturation process, at least someof the additional mutations acquired on the way to our final variant C26can be understood on their basis. The substitution Ile80Thr, which wasrepeatedly found in our screening experiments, occurs at a very criticalposition between Leu79 and Met81 mentioned above. Its side chain isdisplayed on the outside of the β-barrel and seems to be slightlyshifted compared with the original residue in hNGAL and, thus, itsreplacement may affect a local change in the backbone geometry. Thesubstitution Ser127Gln occurs at the tip of loop #4 and is quite remotefrom the bound ligand, which is in agreement with its marginal effect onthe affinity (Table 3). Of the five additional side chain replacementsthat were identified for the variant C26 (42, 48, 49, 55, 75) three arelocated in loop #1, which may lead to a gross conformational change andbring this loop closer to the ligand. The side chain at position 55occurs on the outside of the β-barrel, but, as in the case of position80, an Ile residue is exchanged by Thr, which may have a similar effectmediated via the backbone on the neighboring Gln54, which contacts theDPTA ligand. Furthermore, the replacement of Lys75 by Met occurs at thetip of loop #2, at the last position of a stretch of three consecutiveLys residues and may hence influence the interaction with the side chainof the DTPA group.

Example 16 Generation of hNGAL Variants with Affinity toHexachloronorbornene Hapten

The hNGAL library obtained in Example 2 was used to generate muteinshaving affinity to a hexachloronorbonene hapten. In more detail, thehexachloronorbornene hapten used was hexachloronorbornene N—(CH₂)₅—COOHthe synthesis of which was reported by Hilvert et al., J. Am. Chem. Soc.1989, Vol. 111, 9261-9262 (compound 2 in FIG. 1 of Hilvert et al) whichrepresents a transition state analogue for the [4+2] Diels-Alderreaction (see also, Xu et al., Science, 1999, Vol. 286, 2345-2348).Generation of the hNGAL muteins were carried out essentially inaccordance with the experimental procedure as described in Example 3above and muteins with binding activity to the to hexachloronorbornenehapten were isolated and characterized by ELISA. In these ELISAexperiments signals where obtained for selected hNGAL muteins indicatingthat muteins with at least micromolar K_(D) were generated (data notshown). Also this data, even though being preliminary, show thesuitability of the present invention to generate hNGAL muteins withantibody like properties (that means, for example, having bindingaffinity to any chosen hapten) by subjecting only the 12 amino acidresidues 33, 36, 41, 52, 54, 68, 70, 79, 81, 134, 136 and 138 of thelinear polypeptide sequence of hNGAL to mutagenesis.

Example 17 Affinity Maturation of the hNGAL Variant C26 Towards theMetal Chelate Complex, Y•p-NH₂-Bn-CHX-A″-DTPA Via Phage Display andColony Screening

To randomize loop 1 of the hNGAL variant C26 (SEQ ID NO:10), primersNGAL26 (SEQ ID NO:25; 5′-CCC AGG ACT CCA CCT CAG ACC-3′) and L1Mback(SEQ ID NO:26; 5′-TGG GGC TGC ATT CCC TGC-3′) were applied in a PCR withTaq DNA polymerase as above using pNGAL15-C26 plasmid DNA as template. Asecond PCR fragment was generated using primers L1Mfor (SEQ ID NO:27;5′-GCA GGG AAT GCA GCT CCA NNS NNS NNS NNS NNS CTG CTA NNS NNS ACC GCCTAG ACT TAT GAG C-3′) and P6 (SEQ ID NO:16). Both fragments wereassembled using the flanking primers NGAL26 (SEQ ID NO:25) and P6 (SEQID NO:16). The PCR products were subcloned on pNGAL35 for phagemiddisplay selection.

Phage display panning was performed as described in Example 3 but usingcompetitive conditions and the Y•DTPA-RNase target adsorbed toImmunoSticks. After washing 8 times, bound phagemids were incubated inthe presence of a 400 μM solution of the free metal chelate complex,Y•p-NH₂-Bn-CHX-A″-DTPA, as competitor for 30 min in the first cycle, for3 h in the second cycle, and for 24 h in the third cycle, followed byanother three washing steps and, finally, acid elution. After threecycles of phagemid selection, the enriched pool of hNGAL variants wassubcloned and subjected to the colony screening assay as in Example 3,again applying competitive conditions. Variants giving rise to intensestaining signals in the presence of an approximately thousand-fold molarconcentration of the unlabelled ligand were sequenced. In this mannerthe variant L1 (SEQ ID NO:28) was isolated, which exhibits sixadditional mutations as described in Table 4(R43P/E44V/K46P/D47E/K50L/M51L).

The new hNGAL variant L1 was analyzed by SPR as in Example 8 using aBiacore CM-5 chip with the covalently attached Y-DTPA-RNaseA conjugateand applying the purified recombinant protein. The measured dissociationrate constant of 2.11×10⁻⁴ s⁻¹ was improved by 3.4-fold, compared withthe hNGAL variant C26, while the measured association rate constant of2.96×10⁵ M⁻¹ s⁻¹ was reduced by 10-fold, resulting in a K_(D) of 713 pM.

Another randomization of the hNGAL variants was then performed byerror-prone PCR using the plasmid DNA encoding C26 and L1, respectively,as template and primers P5 (SEQ ID NO:15) and P6 (SEQ ID NO:16) in thepresence of 50 μM dPTP, 50 μM 8-oxo-dGTP, and 1 unit of 9° Nm DNApolymerase, followed by reamplification as described in Example 4. A 1:1mixed phage display library with respect to the two templates wasprepared and used for panning against the Y•DTPA-RNase target as aboveunder competitive conditions. After three cycles of phagemid selection,the enriched pool of hNGAL variants was subcloned and subjected to thecolony screening assay, again applying competitive conditions. 6variants giving rise to intense staining signals were sequenced. All ofthese variants apparently were direct derivatives of L1 but not of C26.Among those, clone CL31 (SEQ ID NO:31) was isolated as the mostpromising hNGAL variant, exhibiting six additional mutations comparedwith L1 as listed in Table 4 (V44M/N65D/G86S/S87P/S99N/L107F).

After preparation of the corresponding soluble protein and SPR analysis,the variant CL31 revealed clearly improved parameters with anassociation rate constant of 4.46×10⁵ M⁻¹ s⁻¹, a dissociation rateconstant of 1.06×10⁻⁴ s⁻¹, and a 237 pM affinity to the metal chelateligand (FIG. 2E). Thus, after complex formation CL31 shows asignificantly longer half life of dissociation (ca. 1.8 h) over theparental lipocalin mutein C26 (16 min). The other selected variantsshowed a similar binding activity (data not shown).

TABLE 4 Amino acid positions of selected hNGAL variants that differ from thesequence of C26. C26 carries additional substitutions compared withwild-type hNGAL as detailed in Table 2. C26 L1 CL2 CL27 CL31 CL34 CL63CL97 Residue (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID No.^(a) NO: 10) NO: 28) NO: 29) NO: 30) NO: 31) NO: 32) NO: 33)NO: 34) 43 Arg Pro   Pro Pro Pro Pro Pro Pro 44 Glu Val Val Val Met ValVal Val 46 Lys Pro Pro Pro Pro Pro Pro Pro 47 Asp Glu Glu Glu Glu GluGlu Glu 50 Lys Leu Leu Leu Leu Leu Leu Leu 51 Met Leu Leu Leu Leu LeuLeu Leu 59 Lys Lys Lys Lys Lys Arg Lys Lys 65 Asn Asn Asp Asp Asp AspAsp Asn 78 Tyr Tyr Tyr Tyr Tyr Tyr Tyr His 86 Gly Gly Gly Ser Ser GlyGly Gly 87 Ser Ser Ser Ser Pro Ser Phe Ser 98 Lys Lys Glu Lys Lys LysLys Lys 99 Ser Ser Ser Ser Asn Ser Ser Ser 103 Leu Leu Leu Leu Leu LeuLeu Ile 107 Leu Leu Leu Leu Phe Leu Leu Phe 110 Val Val Met Val Val ValVal Val 111 Val Val Val Val Val Val Val Ala ^(a)Sequential numbering ofthe mature protein sequence (cf. SwissProt entry P80188).

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including”, “containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention. Theinvention has been described broadly and generically herein. Each of thenarrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group. Further embodiments of the invention willbecome apparent from the following claims.

1. A mutein derived human neutrophil gelatinase-associated lipocalin(hNGAL) comprising at least 3 mutated amino acid residues at any of thesequence positions corresponding to the sequence positions 33, 36, 41,52, 54, 68, 70, 79, 81, 134, 136 and 138 of the linear polypeptidesequence of hNGAL (SEQ ID NO: 1), wherein the mutein binds a giventarget with detectable affinity.
 2. The mutein of claim 1, which has anamino acid sequence further differing from the linear polypeptidesequence of hNGAL (SEQ ID NO: 1) by at least one mutated amino acidresidue at at least one of the sequence positions corresponding to thesequence positions 42, 43, 44, 46, 47, 48, 49, 50, 51, 55, 59, 65, 71,73, 74, 75, 77, 78, 80, 86, 87, 98, 99, 103, 107, 110, 111, 116, 125,127 and 135 of the linear polypeptide sequence of hNGAL.
 3. The muteinof claim 1, wherein the mutein comprises at least 4 mutated amino acidresidues at at least one of the sequence positions corresponding to thesequence positions 33, 36, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52,54, 55, 59, 65, 68, 70, 75, 77, 78, 79, 80, 81, 86, 87, 98, 99, 103,107, 110, 111, 127, 134, 136 and 138 of the linear polypeptide sequenceof hNGAL.
 4. The mutein of claim 1, wherein a Cys residue is introducedat at least one of the sequence positions that correspond to sequencepositions 14, 21, 60, 84, 88, 116, 141, 145, 143, 146 or 158 of the wildtype sequence of hNGAL.
 5. The mutein of claim 1, wherein the muteincomprises with respect to the linear polypeptide sequence of hNGAL atleast one amino acid replacements selected from the group consisting ofGlu28→His; Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro; Arg43→Pro;Glu44→Val; Glu44→Met; Lys46→Pro; Asp47→Glu; Pro48→Leu; Gln49→Leu;Lys50→Leu; Met51→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr; of Lys59→Arg;Asn65→Asp; Ser68→Ala; Leu70→Arg; Lys75→Met; Asp77→Glu; Tyr78→His;Trp79→Ala; Trp79→Leu; Ile80→Thr; Leu; Arg81→Met; Gly86→Ser; Ser87→Pro;Ser87→Phe; Cys87→Ser; Lys98→Glu; Ser99→Asn; Leu103→Ile; Leu107→Phe;Val110→Met; Val111→Ala; Ser127→Gln; Lys134→Ser; Thr136→Ser; Tyr138→Leuand Thr145→Ala.
 6. The mutein of claim 1, wherein the mutein compriseswith respect to the linear polypeptide sequence of hNGAL any one of thefollowing amino acid replacements: (a). Val33→Gln; Leu36→Arg; Ile41→Ala;Tyr52→Thr; Thr54→Gln; Ser68→Ala; Leu70→Arg; Trp79→Ala; Ile 80→Thr;Arg81→Met; Lys134→Ser; and Tyr138→Leu; (b). Val33→Gln; Leu36→Arg;Ile41→Ala; Tyr52→Thr; Thr54→Gln; Ser68→Ala; Leu70→Arg; Trp79→Leu; Ile80→Thr; Arg81→Met; Lys134→Ser; and Tyr138→Leu; (c). Val33→Gln;Leu36→Arg; Ile41→Ala; Tyr52→Thr; Thr54→Gln; Ser68→Ala; Leu70→Arg;Trp79→Leu; Ile 80→Thr; Arg81→Met; Ser127→Gln; Lys134→Ser; andTyr138→Leu; (d). Val33→Gln; Leu36→Arg; Ile41→Ala; Tyr52→Thr; Thr54→Gln;Ser68→Ala; Leu70→Arg; Trp79→Leu; Ile 80→Thr; Arg81→Met; Lys134→Ser;Thr136→Ser; and Tyr138→Leu; (e). Val33→Gln; Leu36→Arg; Ile41→Ala;Tyr52→Thr; Thr54→Gln; Ser68→Ala; Leu70→Arg; Trp79→Leu; Ile 80→Thr;Arg81→Met; Ser127→Gln; Lys134→Ser; Thr136→Ser; and Tyr138→Leu; (f).Val33→Gln; Leu36→Arg; Ile41→Ala; Tyr52→Thr; Thr54→Gln; Ser68→Ala;Leu70→Arg; Asp77→Glu; Trp79→Leu; Ile 80→Thr; Arg81→Met; Ser127→Gln;Lys134→Ser; Thr136→Ser; and Tyr138→Leu; (g). Val33→Gln; Leu36→Arg;Ile41→Ala; Leu42→Pro; Pro48→Leu; Gln49→Leu; Tyr52→Thr; Thr54→Gln;Ile55→Thr; Ser68→Ala; Leu70→Arg; Lys75→Met; Asp77→Glu; Trp79→Leu; Ile80→Thr; Arg81→Met; Ser127→Gln; Lys134→Ser; Thr136→Ser; and Tyr138→Leu;(h). Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro; Arg43→Pro; Glu44→Val;Lys46→Pro; Asp47→Glu; Pro48→Leu; Gln49→Leu; Lys50→Leu; Met51→Leu;Tyr52→Thr; Thr54→Gln; Ile55→Thr; Ser68→Ala; Leu70→Arg; Lys75→Met;Asp77→Glu; Trp79→Leu; Ile 80→Thr; Arg81→Met; Ser127→Gln; Lys134→Ser;Thr136→Ser; and Tyr138→Leu; (i). Val33→Gln; Leu36→Arg; Ile41→Ala;Leu42→Pro; Arg43→Pro; Glu44→Val; Lys46→Pro; Asp47→Glu; Pro48→Leu;Gln49→Leu; Lys50→Leu; Met51→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr;Asn65→Asp; Ser68→Ala; Leu70→Arg; Lys75→Met; Asp77→Glu; Trp79→Leu; Ile80→Thr; Arg81→Met; Lys98→Glu; Val 110→Met; Ser127→Gln; Lys134→Ser;Thr136→Ser; and Tyr138→Leu; (j). Val33→Gln; Leu36→Arg; Ile41→Ala;Leu42→Pro; Arg43→Pro; Glu44→Val; Lys46→Pro; Asp47→Glu; Pro48→Leu;Gln49→Leu; Lys50→Leu; Met51→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr;Asn65→Asp; Ser68→Ala; Leu70→Arg; Lys75→Met; Asp77→Glu; Trp79→Leu; Ile80→Thr; Arg81→Met; Gly86→Ser; Ser127→Gln; Lys134→Ser; Thr136→Ser; andTyr138→Leu; (k). Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro; Arg43→Pro;Glu44→Met; Lys46→Pro; Asp47→Glu; Pro48→Leu; Gln49→Leu; Lys50→Leu;Met51→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr; Asn65→Asp; Ser68→Ala;Leu70→Arg; Lys75→Met; Asp77→Glu; Trp79→Leu; Ile 80→Thr; Arg81→Met;Gly86→Ser; Ser87→Pro; Ser99→Asn; Leu107→Phe; Ser127→Gln; Lys134→Ser;Thr136→Ser; and Tyr138→Leu; (l). Val33→Gln; Leu36→Arg; Ile41→Ala;Leu42→Pro; Arg43→Pro; Glu44→Val; Lys46→Pro; Asp47→Glu; Pro48→Leu;Gln49→Leu; Lys50→Leu; Met51→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr;Lys59→Arg; Asn65→Asp; Ser68→Ala; Leu70→Arg; Lys75→Met; Asp77→Glu;Trp79→Leu; Ile 80→Thr; Arg81→Met; Ser127→Gln; Lys134→Ser; Thr136→Ser;and Tyr138→Leu; (m). Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro;Arg43→Pro; Glu44→Val; Lys46→Pro; Asp47→Glu; Pro48→Leu; Gln49→Leu;Lys50→Leu; Met51→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr; Asn65→Asp;Ser68→Ala; Leu70→Arg; Lys75→Met; Asp77→Glu; Trp79→Leu; Ile 80→Thr;Arg81→Met; Ser87→Phe; Ser127→Gln; Lys134→Ser; Thr136→Ser; andTyr138→Leu; or (n). Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro;Arg43→Pro; Glu44→Val; Lys46→Pro; Asp47→Glu; Pro48→Leu; Gln49→Leu;Lys50→Leu; Met51→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr; Ser68→Ala;Leu70→Arg; Lys75→Met; Asp77→Glu; Tyr78→His; Trp79→Leu; Ile 80→Thr;Arg81→Met; Leu103→Ile; Leu107→Phe; Val111→Ala; Ser127→Gln; Lys134→Ser;Thr136→Ser; and Tyr138→Leu.
 7. The mutein of claim 1, wherein the muteinhas an amino acid sequence selected from the group consisting of theamino acid sequences set forth in SEQ ID NOs: 2-10 and 28-34.
 8. Themutein of claim 1, wherein the mutein binds a small organic molecule ora peptide.
 9. The mutein of claim 1, wherein the mutein is conjugated totargeting moiety with binding affinity for a chosen target molecule,wherein said targeting moiety targets a specific body region in amammal.
 10. The mutein of claim 1, wherein the mutein is conjugated to alabel selected from the group consisting of organic molecules, enzymelabels, radioactive labels, colored labels, fluorescent labels,chromogenic labels, luminescent labels, haptens, digoxigenin, biotin,metal complexes, metals, and colloidal gold.
 11. The mutein of claim 1,wherein the mutein is conjugated to a moiety that extends the serumhalf-life of the mutein.
 12. The mutein of claim 11, wherein the moietythat extends the serum half-life is selected from the group consistingof a polyalkylene glycol molecule, hydroxyethyl starch, a Fc part of animmunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of animmunoglobulin, an albumin binding peptide, and an albumin bindingprotein.
 13. The mutein of claim 1, wherein the mutein is fused at itsN-terminus and/or its C-terminus to a protein, a protein domain or apeptide.
 14. An isolated nucleic acid molecule encoding a mutein ofclaim
 1. 15. The isolated nucleic acid molecule of claim 14 comprised ina phagemid vector.
 16. An isolated cell containing the nucleic acidmolecule of claim
 14. 17. A method for the generation of a mutein ofclaim 1, comprising: (a) subjecting a nucleic acid molecule encoding anhNGAL protein to mutagenesis at a nucleotide triplet coding for at least3 of the sequence positions corresponding to the sequence positions 33,36, 41, 52, 54, 68, 70, 79, 81, 134, 136, and 138 of the linearpolypeptide sequence of hNGAL (SEQ ID NO: 1), resulting in one or moremutein nucleic acid molecule(s), (b) expressing the one more muteinnucleic acid molecule(s) obtained in (a) in a suitable expressionsystem, and (c) identifying one or mutein nucleic acid moleculesencoding a mutein having a detectable binding affinity for a giventarget by means of selection and/or isolation.
 18. The mutein of claim1, which is recombinantly expressed.
 19. A pharmaceutical compositioncomprising the mutein of claim 1 and a pharmaceutically acceptableexcipient.
 20. A diagnostic or analytical kit comprising the mutein ofclaim 1 in a container, optionally containing instructions for its use.